Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling of apoptosis induced by heat stress in U937 human lymphoma cells

ABSTRACT: Hyperthermia is widely used to treat patients with various cancers. 42.5˚C is well known as the inflection point of hyperthermia and generally up to 42˚C of hyperthermia is used in clinical cases combined with other therapies. Here, the effects of heat stress at 42 or 44˚C for 15 min on the gene expression in human lymphoma U937 cells were investigated using an Affymetrix GeneChip system. The cells were treated with heat stress (42 or 44°C for 15 min), followed by incubation for 0, 1, 3 or 6 h at 37°C. The percentage of DNA fragmentation was 8.4 ± 2.2 (mean ± SD) at 42°C for 6 h and 21.0 ± 2.0 at 44°C for 6 h. Of approximately 47,000 probe sets analyzed, many genes that were differentially expressed by a factor 2.0 or greater were identified in the cells treated with heat stress at 42 and 44°C. U937 cells, a human lymphoma cell line, were treated with heat stress (42 or 44°C for 15 min), followed by incubation for 0, 1, 3 or 6 h at 37°C. Non-treated cells served as the control. Total RNA samples were prepared from the cells. Gene expression was analyzed by an Affymetrix GeneChip® system with a Human Genome U133-plus 2.0 array for analysis of over 47,000 transcripts. Sample preparation for array hybridization was carried out as described in the manufacturer’s instruction

ORGANISM(S): Homo sapiens

SUBMITTER: Yoshiaki Tabuchi

PROVIDER: E-GEOD-23405 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:The potential for epigenetic changes in host cells following microbial infection has been widely suggested, but few examples have been reported. We assessed genome-wide patterns of DNA methylation in human macrophage-like U937 cells following infection with Burkholderia pseudomallei, an intracellular bacterial pathogen and the causative agent of human melioidosis. Our analyses revealed significant changes in host cell DNA methylation, at multiple CpG sites in the host cell genome, following infection. Infection induced differentially methylated probes (iDMPs) showing the greatest changes in DNA methylation were found to be in the vicinity of genes involved in inflammatory responses, intracellular signalling, apoptosis and pathogen-induced signalling. A comparison of our data with reported methylome changes in cells infected with M. tuberculosis revealed commonality of differentially methylated genes, including genes involved in T cell responses (BCL11B, FOXO1, KIF13B, PAWR, SOX4, SYK), actin cytoskeleton organisation (ACTR3, CDC42BPA, DTNBP1, FERMT2, PRKCZ, RAC1), and cytokine production (FOXP1, IRF8, MR1). Overall our findings show that pathogenic-specific and pathogen-common changes in the methylome occur following infection. The human leukemic monocyte lymphoma cell line (U937, ATCC CRL-1593.2) was maintained in RPMI 1640 supplemented with 10% foetal bovine serum (FBS) at 37Â°C. U937 cells were differentiated to macrophage-like cells following exposure to 20 ng/ml (final concentration) of phorbol 12-myristate 13-acetate (PMA) for 48 hours at 37Â°C and differentiation evidenced by increased adherence to tissue culture flasks. Overnight cultures of B. pseudomallei K96243 were diluted in L-15 medium and added to differentiated U937 cells at a multiplicity of infection (MOI) of 10. Uninfected controls were overlaid with L15 medium only. The cells were then incubated at 37Â°C to allow infection. The cells were washed 3 times with PBS and incubated with fresh L15 medium containing 1mg/ml kanamycin to kill extracellular bacteria. At appropriate time points the cells were washed 3 times in warm PBS and lysed with 0.1% (vol/vol) Triton X-100. DNA was isolated using an AllPrep kit (Qiagen) and stored at -80ÂºC until required. DNA yield was measured using a Nanodrop instrument with measurements between 22.8 â 50.6 ng/ul. Two experiments were carried out using the above procedure. In Experiment 1, DNA was collected from uninfected and infected cells at 2 hours (T2), and 4 hours (T4) post infection (2 biological replicates and 2 technical replicates from each group). In Experiment 2, DNA was collected from uninfected and infected cells at 1 hour (T1), 2 hours (T2), 3 hours (T3) and 4 hours (T4) post infection (1 sample from each group). The DNA methylation profile was determined using the Infinium HumanMethylation450 BeadChip (450K) (Illumina Inc.) following the manufacturerâs instructions. The data was extracted and the initial analysis was performed using GenomeStudio (2010.3) methylation module (1.8.5). Quality control checks and quantile normalisation were implemented using WateRmelon. Samples with more than 1% of sites with a detection p-value greater than 0.05 were removed as were probes with 1% of samples with a detection p-value greater than 0.05. Probes were removed if they had a bead count less than 3 in 1% of samples. Cross-hybridizing probes were removed, leaving 425496 probes for analysis. Here we report DNA methylation profile of 18 samples (10 infected and 8 control).

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Transcription profiling of apoptosis induced by heat stress in U937 human lymphoma cells

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