Transcription profiling of human colorectal carcinoma cell lines HCT116 and HT29 treated with 5-ASA
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
SUBMITTER: Petr Dobes
PROVIDER: E-TABM-768 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:In the present study we aimed to develop a custom made cDNA microarray, the CodStress array, for Atlantic cod to be used as a diagnostic tool in environmental monitoring of polluted waters. We wanted to investigate if the gene expression profiles would reflect environmental levels and composition of pollutants in two saltwater recipients, S?rfjorden and Store Lungeg?rdsvann.
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Project description:Understanding pathogen recognition and mechanisms in Atlantic cod are of significant importance for both basic research on wild populations and health management in aquaculture. A microarray approach was utilized to search for effects of viral (poly I:C/ polyinosinic acid:polycytidylic acid), bacterial (LPS/lipopolysaccharide) and polyclonal activator (PHA-L/phytohaemoagglutinin) stress in Atlantic cod head kidney cells. LPS cell activation increased mRNA expression of chemokine/Interleukin 8 (CXCL8/IL-8); interleukin -1? (IL-1?); cyclooxygenase 2 (COX2); leukocyte derived chemotaxin 2 (LECT2); LOC100698154, encoding a protein with unknown function; carboxyl-esterase 2 (CES2) and environmental biomarker cytochrome P450 1A (CYP1A). Mitogen activated protein kinase p38 (p38MAPK) and cathepsin F (CTSF) were downregulated by LPS. The antiviral responses induced by double stranded RNA (Poly I:C) clearly increased transcription of Toll like receptor 3 (TLR3) and interferon stimulating gene 15 (ISG15). The PHA response seemed to be more non-specific. Special for the PHA induction were the increase in Major histocompatibility complex class I (MHCI). CC chemokine type 2 (CK2) mRNA expression was increased by PHA, LPS and poly I:C, while p38MAPK and LECT2 was downregulated by PHA. Oxidative stress related genes like catalase and glutaredoxin (GLRX2) and the anti-apoptotic gene Bcl-2 showed no transcriptional changes compared to control in any of the treatments. Especially Poly I:C, but also LPS, induced leukotriene B4 (LTB4) and leukotriene B5 (LTB5) synthesis, while small amounts of prostaglandine E2 (PGE2) seemed to be constitutively produced in untreated cells, a production that was slightly elevated when exposing cells for LPS. This study reveals distinct signatures of bacteria and virus transcriptional responses in cod head kidney cells. In addition, the novel finding that Cyp1a was upregulated during the antibacterial response indicates a connection between immunity and aryl hydrocarbon receptor (AhR) activation in Atlantic cod.
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Project description:Developments in mass spectrometry (MS)-based analyses of glycoproteins have been important to study changes in glycosylation related to disease. Recently, the characteristic pattern of oxonium ions in glycopeptide fragmentation spectra had been used to assign different sets of glycopeptides. In particular this was helpful to discriminate between O-GalNAc and O-GlcNAc. Here, we thought to investigate how such information can be used to examine quantitiative proteomics data. For this purpose, we used TMT (Tandem Mass Tag)-labeled samples from total cell lysates and secreted proteins from three different colorectal cancer cell lines. Following automated glycopeptide assignment (Byonic) and evaluation of the presence and relative intensity of oxonium ions, we observed that in particular the ratio of the ions at m/z 144.066 and 138.055, respectively, could be used to discriminate between O-GlcNAcylated and O-GalNAcylated peptides, with concomittant relative quantification between the different cell lines. Among the O-GalNAcylated proteins, we also observed anterior gradient protein 2 (AGR2), a protein which glycosylation site and status was hitherto not well documented. Using a combination of multiple fragmentation methods we then not only assigned the site of modification, but also showed different glycosylation between intracellular (ER-resident) and secreted AGR2. Overall, our study shows the potential of broad application of the use of the relative intensities of oxonium ions for the confident assignment of glycopeptides, even in complex proteomics datasets.
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