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Toxicology and Applied Pharmacology Oct 2023Modern toxicology's throughput has dramatically increased due to alternative models, laboratory automation, and machine learning. This has enabled comparative studies...
Modern toxicology's throughput has dramatically increased due to alternative models, laboratory automation, and machine learning. This has enabled comparative studies across species and assays to prioritize chemical hazard potential and to understand how different model systems might complement one another. However, such comparative studies of high-throughput data are still in their infancy, with more groundwork needed to firmly establish the approach. Therefore, this study aimed to compare the bioactivity of the NIEHS Division of Translational Toxicology's (DTT) 87-compound developmental neurotoxicant (DNT) library in zebrafish and an in vitro high-throughput cell culture system. The early life-stage zebrafish provided a whole animal approach to developmental toxicity assessment. Chemical hits for abnormalities in embryonic zebrafish morphology, mortality, and behavior (ZBEscreen™) were compared with chemicals classified as high-risk by the Cell Health Index (CHI™), which is an outcome class probability from a machine learning classifier using 12 parameters from the SYSTEMETRIC® Cell Health Screen (CHS). The CHS was developed to assess human toxicity risk using supervised machine learning to classify acute cell stress phenotypes in a human leukemia cell line (HL60 cells) following a 4-h exposure to a chemical of interest. Due to the design of the screen, the zebrafish assays were more exhaustive, yielding 86 total bioactive hits, whereas the SYSTEMETRIC® CHS focusing on acute toxicity identified 20 chemicals as potentially toxic. The zebrafish embryonic and larval photomotor response assays (EPR and LPR, respectively) detected 40 of the 47 chemicals not found by the zebrafish morphological screen and CHS. Collectively, these results illustrate the advantages of using two alternative models in tandem for rapid hazard assessment and chemical prioritization and the effectiveness of CHI™ in identifying toxicity within a single multiparametric assay.
Topics: Animals; Humans; Zebrafish; Biological Assay; HL-60 Cells; Larva; Leukemia
PubMed: 37604412
DOI: 10.1016/j.taap.2023.116659 -
Indian Journal of Pathology &... Apr 2024
Topics: Humans; Inclusion Bodies; Granulocyte Precursor Cells; Microscopy; Male; Bone Marrow; Histocytochemistry; Female
PubMed: 38391365
DOI: 10.4103/ijpm.ijpm_33_22 -
Toxins Apr 2022Cry41Aa, also called parasporin-3, belongs to a group of toxins from the entomopathogenic bacterium that show activity against human cancer cells. Cry41Aa exhibits...
Cry41Aa, also called parasporin-3, belongs to a group of toxins from the entomopathogenic bacterium that show activity against human cancer cells. Cry41Aa exhibits preferential cytocidal activity towards HL-60 (human promyelocytic leukaemia cells) and HepG2 (human liver cancer cells) cell lines after being proteolytically activated. To better understand the mechanism of action of Cry41Aa, we evolved resistance in HepG2 cells through repeated exposure to increasing doses of the toxin. Concentrations of Cry41Aa that killed over 50% of the parental HepG2 cells had no significant effect on the viability of the resistant cells and did not induce either pore formation or p38 phosphorylation (both characteristic features of pore-forming toxins). Preliminary RNA sequencing data identified AQP9 as a potential mediator of resistance, but extensive investigations failed to show a causal link and did not support an enhanced cell repair process as the resistance mechanism.
Topics: Bacillus thuringiensis; Bacterial Proteins; HL-60 Cells; Hep G2 Cells; Humans
PubMed: 35622566
DOI: 10.3390/toxins14050319 -
The Science of the Total Environment Apr 2023The cellular and molecular mechanisms by which atmospheric pollution from particulate matter and/or electromagnetic fields (EMFs) may prove harmful to human health have...
The HL-60 human promyelocytic cell line constitutes an effective in vitro model for evaluating toxicity, oxidative stress and necrosis/apoptosis after exposure to black carbon particles and 2.45 GHz radio frequency.
The cellular and molecular mechanisms by which atmospheric pollution from particulate matter and/or electromagnetic fields (EMFs) may prove harmful to human health have not been extensively researched. We analyzed whether the combined action of EMFs and black carbon (BC) particles induced cell damage and a pro-apoptotic response in the HL-60 promyelocytic cell line when exposed to 2.45 GHz radio frequency (RF) radiation in a gigahertz transverse electromagnetic (GTEM) chamber at sub-thermal specific absorption rate (SAR) levels. RF and BC induced moderately significant levels of cell damage in the first 8 or 24 h for all exposure times/doses and much greater damage after 48 h irradiation and the higher dose of BC. We observed a clear antiproliferative effect that increased with RF exposure time and BC dose. Oxidative stress or ROS production increased with time (24 or 48 h of radiation), BC dose and the combination of both. Significant differences between the proportion of damaged and healthy cells were observed in all groups. Both radiation and BC participated separately and jointly in triggering necrosis and apoptosis in a programmed way. Oxidative-antioxidant action activated mitochondrial anti-apoptotic BCL2a gene expression after 24 h irradiation and exposure to BC. After irradiation of the cells for 48 h, expression of FASR cell death receptors was activated, precipitating the onset of pro-apoptotic phenomena and expression and intracellular activity of caspase-3 in the mitochondrial pathways, all of which can lead to cell death. Our results indicate that the interaction between BC and RF modifies the immune response in the human promyelocytic cell line and that these cells had two fates mediated by different pathways: necrosis and mitochondria-caspase dependent apoptosis. The findings may be important in regard to antimicrobial, inflammatory and autoimmune responses in humans.
Topics: Humans; HL-60 Cells; Apoptosis; Necrosis; Radio Waves; Oxidative Stress; Carbon; Electromagnetic Fields
PubMed: 36632900
DOI: 10.1016/j.scitotenv.2023.161475 -
JCI Insight May 2018Optimal management of acute myeloid leukemia (AML) requires monitoring of treatment response, but minimal residual disease (MRD) may escape detection. We sought to...
BACKGROUND
Optimal management of acute myeloid leukemia (AML) requires monitoring of treatment response, but minimal residual disease (MRD) may escape detection. We sought to identify distinctive features of AML cells for universal MRD monitoring.
METHODS
We compared genome-wide gene expression of AML cells from 157 patients with that of normal myeloblasts. Markers encoded by aberrantly expressed genes, including some previously associated with leukemia stem cells, were studied by flow cytometry in 240 patients with AML and in nonleukemic myeloblasts from 63 bone marrow samples.
RESULTS
Twenty-two (CD9, CD18, CD25, CD32, CD44, CD47, CD52, CD54, CD59, CD64, CD68, CD86, CD93, CD96, CD97, CD99, CD123, CD200, CD300a/c, CD366, CD371, and CX3CR1) markers were aberrantly expressed in AML. Leukemia-associated profiles defined by these markers extended to immature CD34+CD38- AML cells; expression remained stable during treatment. The markers yielded MRD measurements matching those of standard methods in 208 samples from 52 patients undergoing chemotherapy and revealed otherwise undetectable MRD. They allowed MRD monitoring in 129 consecutive patients, yielding prognostically significant results. Using a machine-learning algorithm to reduce high-dimensional data sets to 2-dimensional data, the markers allowed a clear visualization of MRD and could detect 1 leukemic cell among more than 100,000 normal cells.
CONCLUSION
The markers uncovered in this study allow universal and sensitive monitoring of MRD in AML. In combination with contemporary analytical tools, the markers improve the discrimination between leukemic and normal cells, thus facilitating data interpretation and, hence, the reliability of MRD results.
FUNDING
National Cancer Institute (CA60419 and CA21765); American Lebanese Syrian Associated Charities; National Medical Research Council of Singapore (1299/2011); Viva Foundation for Children with Cancer, Children's Cancer Foundation, Tote Board & Turf Club, and Lee Foundation of Singapore.
Topics: ADP-ribosyl Cyclase 1; Adolescent; Adult; Algorithms; Antigens, CD; Antigens, CD34; Antineoplastic Combined Chemotherapy Protocols; Biomarkers, Tumor; Case-Control Studies; Child; Child, Preschool; Disease Progression; Gene Expression; Gene Expression Profiling; Granulocyte Precursor Cells; Humans; Infant; Leukemia, Myeloid, Acute; Membrane Proteins; Middle Aged; Monitoring, Physiologic; Neoplasm, Residual; Young Adult
PubMed: 29720577
DOI: 10.1172/jci.insight.98561 -
Oncotarget Sep 2016CD81 is a cell surface protein which belongs to the tetraspanin family. While in multiple myeloma its expression on plasma cells is associated with worse prognosis, this...
CD81 is a cell surface protein which belongs to the tetraspanin family. While in multiple myeloma its expression on plasma cells is associated with worse prognosis, this has not yet been explored in acute myeloid leukemia (AML). We measured membrane expression of CD81 on AML cells at diagnosis, evaluated its association with AML characteristics and its influence on patient outcome after intensive chemotherapy in a cohort of 134 patients. CD81 was detected in 92/134 (69%) patients. Patients with AML expressing CD81 had elevated leukocyte count (P=0.02) and were more likely classified as intermediate or adverse-risk by cytogenetics (P<0.001). CD81 expression had a negative impact on survival (event-free survival, overall survival and relapse-free survival) in univariate (P<0.001) and in multivariate analyses (P=0.003, 0.002 and <0.001, respectively). CD81 has a negative impact on OS in patients with NPM1 mutation (P=0.01) and in patients ELN-favorable (P=0.002). In conclusion, this cell surface marker may be a new prognostic marker for diagnostic risk classification and a potential therapeutic target for drug development in AML.
Topics: Adult; Age Factors; Aged; Biomarkers, Tumor; Cell Membrane; Disease-Free Survival; Female; Flow Cytometry; Follow-Up Studies; Granulocyte Precursor Cells; Humans; Kaplan-Meier Estimate; Leukemia, Myeloid, Acute; Leukocyte Count; Male; Middle Aged; Mutation; Nuclear Proteins; Nucleophosmin; Prognosis; Tetraspanin 28; Young Adult
PubMed: 27566555
DOI: 10.18632/oncotarget.11481 -
Cytometry. Part a : the Journal of the... Apr 2006Histone H1 and H3 phosphorylation associated with chromatin condensation during mitosis has been studied extensively. Less is known on histone modifications that occur...
Sequential phosphorylation of Ser-10 on histone H3 and ser-139 on histone H2AX and ATM activation during premature chromosome condensation: relationship to cell-cycle phase and apoptosis.
BACKGROUND
Histone H1 and H3 phosphorylation associated with chromatin condensation during mitosis has been studied extensively. Less is known on histone modifications that occur during premature chromosome condensation (PCC). The aim of the present study was to reveal the status of histone H3 and H2AX phosphorylation on Ser-10 and Ser-139, respectively, as well as ATM activation through phosphorylation on Ser-1981, during PCC, and relate these events to cell-cycle phase and to initiation of apoptosis.
MATERIALS AND METHODS
To induce PCC, A549 and HL-60 cells were exposed to the phosphatase inhibitor calyculin A (Cal A). Phosphorylation of histone H3 and H2AX as well as ATM activation were detected immunocytochemically concurrent with analysis of cellular DNA content and activation of caspase-3, a marker of apoptosis. The intensity of cellular fluorescence was measured by flow- or laser scanning cytometry.
RESULTS
Induction of PCC led to rapid histone H3 phosphorylation, followed by activation of ATM and then H2AX phosphorylation in both, HL-60 and A549 cells. All these events occurred sequentially, prior to caspase-3 activation, and affected cells in all phases of the cell cycle. ATM activation and H2AX phosphorylation was seen during mitosis of A549 but not HL-60 cells.
CONCLUSIONS
Because the Cal A-induced phosphorylation of histone H3 and H2AX, and of ATM, precede caspase-3 activation these modifications are pertinent to PCC and not to apoptosis-associated chromatin condensation. The sequence of histone H3 and H2AX phosphorylation and ATM activation during PCC is compatible with a role of ATM in mediating phosphorylation of H2AX but not H3. Mitosis in some cell types may proceed without ATM activation and H2AX phosphorylation.
Topics: Apoptosis; Ataxia Telangiectasia Mutated Proteins; Cell Cycle; Cell Cycle Proteins; Chromatin Assembly and Disassembly; DNA Damage; DNA Replication; DNA-Binding Proteins; Enzyme Inhibitors; Flow Cytometry; HL-60 Cells; Histones; Humans; Immunohistochemistry; Marine Toxins; Microscopy, Fluorescence; Oxazoles; Phosphorylation; Point Mutation; Protein Serine-Threonine Kinases; Serine; Tumor Suppressor Proteins
PubMed: 16528736
DOI: 10.1002/cyto.a.20257 -
Cytometry. Part B, Clinical Cytometry May 2013T-cell immunoglobulin mucin-3 (TIM3) has recently been described as an acute myeloid leukemia (AML) stem cell antigen expressed on leukemic myeloblasts, but not on...
BACKGROUND
T-cell immunoglobulin mucin-3 (TIM3) has recently been described as an acute myeloid leukemia (AML) stem cell antigen expressed on leukemic myeloblasts, but not on normal hematopoietic stem cells. TIM3 is also expressed by monocytes, natural killer cells, and several T cell subsets; however, normal myeloblasts have not been well-characterized or compared to AML. A specific flow cytometric marker capable of separating leukemic myeloblasts from non-neoplastic myeloblasts would be diagnostically useful, especially in the post-chemotherapy setting.
METHODS
TIM3 myeloblast expression was assessed in 69 bone marrow and/or peripheral blood specimens, including 27 AML and 42 non-neoplastic cases (20 with a recent history of chemotherapy). TIM3 median fluorescence intensity (MFI) was evaluated within myeloblast, monocyte, T cell, and natural killer cell populations.
RESULTS
The median percentage of myeloblasts positive for TIM3 was lower in non-neoplastic specimens without a history of recent chemotherapy (50.3%) as compared to AML (71.4%), but not significantly different as compared to non-leukemic myeloblasts in the post-chemotherapy setting (72.4%). Mean myeloblast TIM3 MFI was higher in AML myeloblasts and non-leukemic myeloblasts in the post-chemotherapy setting as compared to non-neoplastic myeloblasts in cases lacking a history of chemotherapy. Mean monocyte, natural killer cell, and T-cell TIM3 MFI remained relatively constant in varied clinical settings.
CONCLUSIONS
We confirm that leukemic myeloblasts overexpress TIM3 as compared to non-neoplastic controls; however, high levels of expression may also be seen among non-leukemic myeloblasts in the post-chemotherapy setting. This overlap limits the diagnostic utility of TIM3 as a specific marker of neoplasia.
Topics: Antineoplastic Agents; Biomarkers; Bone Marrow; Cell Count; Flow Cytometry; Gene Expression; Granulocyte Precursor Cells; Hepatitis A Virus Cellular Receptor 2; Humans; Killer Cells, Natural; Leukemia, Myeloid, Acute; Membrane Proteins; Monocytes; T-Lymphocytes
PubMed: 23554257
DOI: 10.1002/cyto.b.21080 -
BMC Genomics Aug 2007Human myelopoiesis is an exciting biological model for cellular differentiation since it represents a plastic process where multipotent stem cells gradually limit their...
BACKGROUND
Human myelopoiesis is an exciting biological model for cellular differentiation since it represents a plastic process where multipotent stem cells gradually limit their differentiation potential, generating different precursor cells which finally evolve into distinct terminally differentiated cells. This study aimed at investigating the genomic expression during myeloid differentiation through a computational approach that integrates gene expression profiles with functional information and genome organization.
RESULTS
Gene expression data from 24 experiments for 8 different cell types of the human myelopoietic lineage were used to generate an integrated myelopoiesis dataset of 9,425 genes, each reliably associated to a unique genomic position and chromosomal coordinate. Lists of genes constitutively expressed or silent during myelopoiesis and of genes differentially expressed in commitment phase of myelopoiesis were first identified using a classical data analysis procedure. Then, the genomic distribution of myelopoiesis genes was investigated integrating transcriptional and functional characteristics of genes. This approach allowed identifying specific chromosomal regions significantly highly or weakly expressed, and clusters of differentially expressed genes and of transcripts related to specific functional modules.
CONCLUSION
The analysis of genomic expression during human myelopoiesis using an integrative computational approach allowed discovering important relationships between genomic position, biological function and expression patterns and highlighting chromatin domains, including genes with coordinated expression and lineage-specific functions.
Topics: Antigens, CD34; Cell Differentiation; Cell Lineage; Chromosomes, Human; Cluster Analysis; Computational Biology; Eosinophils; Erythroblasts; Fetal Blood; Gene Expression; Gene Expression Profiling; Genome, Human; Genomics; Granulocyte Precursor Cells; Hematopoietic Stem Cells; Humans; Models, Biological; Monocytes; Myeloid Cells; Myelopoiesis; Neutrophils; Oligonucleotide Array Sequence Analysis; Software
PubMed: 17683550
DOI: 10.1186/1471-2164-8-264 -
Nature Communications May 2017While intron retention (IR) is considered a widely conserved and distinct mechanism of gene expression control, its regulation is poorly understood. Here we show that...
While intron retention (IR) is considered a widely conserved and distinct mechanism of gene expression control, its regulation is poorly understood. Here we show that DNA methylation directly regulates IR. We also find reduced occupancy of MeCP2 near the splice junctions of retained introns, mirroring the reduced DNA methylation at these sites. Accordingly, MeCP2 depletion in tissues and cells enhances IR. By analysing the MeCP2 interactome using mass spectrometry and RNA co-precipitation, we demonstrate that decreased MeCP2 binding near splice junctions facilitates IR via reduced recruitment of splicing factors, including Tra2b, and increased RNA polymerase II stalling. These results suggest an association between IR and a slower rate of transcription elongation, which reflects inefficient splicing factor recruitment. In summary, our results reinforce the interdependency between alternative splicing involving IR and epigenetic controls of gene expression.
Topics: Alternative Splicing; Animals; Cells, Cultured; DNA Methylation; Granulocyte Precursor Cells; Introns; Methyl-CpG-Binding Protein 2; Mice; Protein Binding; RNA Polymerase II; RNA Splice Sites; RNA Splicing Factors
PubMed: 28480880
DOI: 10.1038/ncomms15134