RNA-seq analysis of neutrophils from CD40L-deficient patients and healthy controls, as well as HL-60 promielocytic cell line
ABSTRACT: We aim to characterize to effects of the absence of CD40L on neutrophil transcriptome and the effect of soluble CD40L on HL60 cells. For this purpose Total RNA of isolated neutrophils from three CD40L-deficient patients and three healthy controls as well as HL-60 cells from ATCC (HL-60 (ATCC CCL-240) were analyzed by RNAseq. Before RNA obtention, neutrophils were incubated for 2 hours in the presence or absence of 100 U/ml rhIFN- (Immukine, Boehringer Ingelheim), and HL-60 cells cultured for 6 days in the presence or absence of 500 ng/mL sCD40L and/or 1,0 % dimethyl sulfoxide (DMSO).
Project description:The purpose of the present study was to elucidate the exocytic events in neutrophil-mediated inflammation. Therefore, we first investigated the expression of genes implicated in these exocytic events in a cell model for neutrophils, the promyelocytic HL-60 cell line. By the addition of 1.3% DMSO during 4.5 days, these cells are differentiated into neutrophil-like dHL-60 cells. Non-differentiated cells served as control. Upon cell differentiation, we i) identified differentially expressed genes implicated in the mechanisms of vesicle transport, and ii) found some of them being upregulated. This upregulated gene expression upon DMSO-maturation points towards a functional role in these cells. Secondly, we confirmed the expression of the selected genes in human neutrophils, isolated from venous blood of 4 different donors.
Project description:Transcriptional profiling of human leukemia HL-60 cells comparing ATRA treated HL-60 cells with ATRA plus ATO. Goal was to determine the effects of ATO on ATRA induced differentiation of HL-60 cells. Two-condition experiment, ATRA vs. ATRA plus ATO treated HL-60 cells.
Project description:Curcumin has antileukemic potential that is mediated by dfifferent pathways. Because we could not confirm that the Arylhydrocarbonreceptor, a ligand activated transcription factor, is involved in curcumins antileukemic effects, we performed microarray experiments to have an overall screening of the pathways affected by curcumin in HL-60 cells. We identified an involvement of the vitamin D receptor in the effects of curcumin on HL-60 cells by comparing our results with other experiments. Experiment Overall Design: HL-60 cells were treated for 24h with 100 µM curcumin, 10 nM 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), or 0.5 % DMSO as control.
Project description:To reveal the gene expression profile changes after exposure to BT, one of benzene metabolites, with or without ABAH, a myeloperoxidase (MPO) inhibitor, and to elucidate how MPO is involved in myelotoxicity of BT, we performed microarray analysis. HL-60 cells suspended in RPMI 1640 containing with 10% of heat-inactivated FBS at 4×10^5/ mL were incubated with or without BT (50 μM) at 37℃ for one or 4 hours. For MPO inhibition experiment, HL-60 (4×10^5 cells/ mL) were pretreatment with 100 μM of ABAH in RPMI 1640/10% of heat-inactivated FBS at 37℃ for 24 hours. Media was then replaced with new media containing the regent plus BT (50 μM) and incubate at 37℃ for one or 4 hours. Unexposed HL-60 cells were used as controls. To validate the microarray results, we selected 22 genes and conducted a real-time PCR. Gene expression alteration induced by 50 μM of BT in HL-60 cells, with or without ABAH, was measured at 1 and 4 hours after exposure to it. Three independent experiments were performed at each time (1 or 4 hours).
Project description:The purpose of the present study was to elucidate in more details the molecular mechanisms of neutrophil-mediated inflammation. We therefore investigated the time-dependent stimulatory potential of LPS from Escherichia coli on cytokine response in neutrophil-like HL-60 cells. <br><br>To get a general overview on the total mRNA changes in DMSO-differentiated HL-60 cells, we performed whole-transcript analysis of LPS-stimulated dHL-60 cells after 2h and 6h of LPS treatment. The samples were collected from three independent experiments from three different passages in cell culture. Non-stimulated dHL-60 cells served as control. We identified the differentially expressed cytokine genes implicated in the human inflammatory response and prominent for their role in neutrophil-mediated inflammatory processes.
Project description:GATA2 mutants were discovered in families predisposed to MDS/AML and in sporadic cases of CML-blast crisis. Promyelocytic HL-60 cells were transduced with lentiviral vectors that express GATA2 WT or T354M, 355delT or L359V mutants upon addition of 4-hydroxy tamoxifen (4HT). Microarrays were performed to identify GATA2 WT signatures and differences caused by these mutations. HL-60 cells transduced with GATA2 (WT, T354M, 355delT and L359V) lentiviruses were treated with 4HT for 24 hours and RNA isolated and gene expression measured on Affymetrix microarrays.
Project description:To identify the mechanisms controlling chronic myeloid leukemia (CML) and acute myeloid leukemia (AML) in humans, we analyzed genome-wide transcription dynamics in three myeloid leukemia cell lines (K562, HL-60, and THP1) using high-throughput sequencing technology. Using KEGG analysis, we found that the ERK/MAPK, JAK-STAT and ErbB pathways promoted proliferation and metabolism in CML. However, in AML, differentiation and apoptosis blocking resulted in the accumulation of blast cells in marrow. In addition, each cell type had unique characteristics. K562 cells are an ideal model for studying erythroid differentiation and globin gene expression. The chemokine signaling pathway and Fc gamma R-mediated phagocytosis were markedly upregulated in HL-60 cells. In THP1 cells, highly expressed genes ensured strong phagocytosis by monocytes. Further, we provide a new insight into myeloid development. The abundant data sets and well-defined analysis methods will provide a resource and strategy for further investigation of myeloid leukemia. Compare mRNA transcriptomes of three different cell lines
Project description:HL-60 cells were treated for 48hr with lithium, sb415286 or GS87 and total RNA was prepared using Trizol (Invitrogen). Next cRNA was prepared using the targetAMP Nano Kit (Epicentre). Finally, the cRNA was hybridized to the HT-12 Expression Chip (Illumina) and gene expression of the samples was determined.
Project description:Daunorubicin (DNR) and Cytarabin (Ara-C) are the main chemotherapeutic drugs used for Acute Myeloid Leukemias (AML) treatment. However, their mode of action is not well understood. To decipher if these drug would induce rapid transcriptional reprogramming, we treated HL-60 AML cell line with DNR or Ara-C for 3 hours and carried out a transcriptomic analyses. In addition, we had shown that Reactive Oxigen Species (ROS) produced by NADPH oxidases (NOX) are involved in DNR-induced gene expression. We therefore also performed a transcriptomic analyses in HL-60 cells treated with DNR and VAS2870, an NADPH oxidase inhibitor. HL-60 cells were treated or not for 3 hours with 2 µM Ara-C, 1 µM DNR or 1 µM DNR + 20µM VAS 2870. RNAs were purified from 3 independent experiments and used to probe Affymetrix Human Gene 2.0 ST Genechips