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Systems biology of gene expression of bladder papilloma cells modulated by a malignant-derived extracellular matrix

ABSTRACT: This study was undertaken to identify how gene expression of the RT4 bladder papilloma cells respond to extracellular matrics (ECM) Matrigel. Gene expression profile of RT4 cells grown on Plastic was compared with expression profiles of RT4 cells grown on Matriges over 12 hours-9 days timecourse. Total 5881 hypervariable (HV) genes were found to vary significantly over the time points, indicating a response to matrix change. Correlational clustering showed these HV genes fell into distinct families, predominantly genes which became highly expressed on ECM. Another cluster show genes expressed at 12 hours after grown on Matrigel and third cluster show genes whose expression decreased below noise level on Matrigel. This indicate that processes responsible for cell adaptation to ECM happen within first 12 hours and the second cluster form last response of those active processes. Over 1-9 days of cell grows on Matrigel those genes are constantly expressed. Cell signaling and cellular growth and proliferation accounted for major functions for those genes. G-protein signaling, immune response and NF-kB signaling accounted for most dramatically affected canonical pathways. Genes sharing similar ontologies also share several overrepresented transcription regulatory elements confirmed by independent experiment on Panomics platform.Thus, rapid response for ECM orchestrated by several transcription factors was observed. Identified canonical pathways indicate possible targets for manipulating cell phenotype on ECM. Keywords: extracellular matrix, gene expression, RT4, Matrigel, microarray, time course

ORGANISM(S): Homo sapiens

PROVIDER: GSE9291 | GEO | 2008/06/10

SECONDARY ACCESSION(S): PRJNA102931

REPOSITORIES: GEO

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Project description:This study was undertaken to identify how gene expression of the RT4 bladder papilloma cells respond to extracellular matrics (ECM) Matrigel. Gene expression profile of RT4 cells grown on Plastic was compared with expression profiles of RT4 cells grown on Matriges over 12 hours-9 days timecourse. Total 5881 hypervariable (HV) genes were found to vary significantly over the time points, indicating a response to matrix change. Correlational clustering showed these HV genes fell into distinct families, predominantly genes which became highly expressed on ECM. Another cluster show genes expressed at 12 hours after grown on Matrigel and third cluster show genes whose expression decreased below noise level on Matrigel. This indicate that processes responsible for cell adaptation to ECM happen within first 12 hours and the second cluster form last response of those active processes. Over 1-9 days of cell grows on Matrigel those genes are constantly expressed. Cell signaling and cellular growth and proliferation accounted for major functions for those genes. G-protein signaling, immune response and NF-kB signaling accounted for most dramatically affected canonical pathways. Genes sharing similar ontologies also share several overrepresented transcription regulatory elements confirmed by independent experiment on Panomics platform.Thus, rapid response for ECM orchestrated by several transcription factors was observed. Identified canonical pathways indicate possible targets for manipulating cell phenotype on ECM. Keywords: extracellular matrix, gene expression, RT4, Matrigel, microarray, time course Three dimensional gel cultures with Matrigel were performed by layering 0.8 mls of ice cold Matrigel onto polyethylene terephthalate membranes of 6-well cell culture inserts (Falcon, Becton-Dickinson Labware, Franklin Lakes, NJ). Gels were solidified at 37Âº C. RT4 cells were trypsinized, brought up on 20 mls McCoys media with 10% Fetal Calf Serum (FCS). Cells were centrifuged then brought up in McCoys media with 10% FCS to a concentration of 500,000 cells/250Âµl. 250Âµl added on top of each solidified matrigel disc and 2mls of McCoys with 10% FCS layered underneath each culture insert. Cells were maintained for 10 days and media replenished underneath the insert twice a week. Cells were harvested at 12 hrs, 24hrs, 2 days, 3 days, 4days, 6 days, 7 days, 8 days, and 9 days of growth in culture using Matrisperse (Falcon, Becton-Dickinson Labware, Franklin Lakes, NJ) and RNA collected. Data were normalized as was described previously in Dozmorov I et al., Nucleic Acids Res. 2004 Oct 28;32(19). In general, intensities were converted to expression and normalized to low-expressed genes, which provide possible technical noise level. The arrays are then adjusted to each other by robust linear regression and this Log10 transformed data presented for each sample. Group of low-expressed genes is selected by normal distribution fitting. This group provides internal standard of measurement noise (ISMN). Genes above 5SD from mean of ISMN were selected as expressed

Project description:This study corresponds to a re-analysis and meta-analysis of the five transcriptomic studies related to spontaneous tolerance to renal allograft in human. It examines the transcriptome of peripheral blood samples from six distinct clinical groups: healthy volunteers (HV); tolerant patients (TOL); stable patients on minimal immunosuppression (MIS) or maintained on classical immunosuppressive therapy (STA); patients showing either signs of chronic rejection (CR) or acute rejection (AR). The initial studies are the following: -Study 1 from Braud C. et al. (J Cell Biochem., 2008) published under GEO accession number GSE47755 and comprising 250 unique samples (8 HV; 21 TOL; 190 STA; 31 CR). -Study 2 from Brouard S. et al. (Proc Natl Acad Sci U S A., 2007) published under GEO accession number GSE47683 and comprising 67 unique samples (8 HV; 12 TOL; 10 MIS; 12 STA; 11 CR; 14 AR). - Study 3 from Lozano JJ. et al. (Am J Transplant., 2011) published under GEO accession number GSE22707 and comprising 42 unique samples (6 HV; 12 TOL; 12 STA; 12 CR). - Study 4 from Newell KA. et al. (J Clin Invest., 2010) published under GEO accession number GSE22229 and comprising 58 unique samples (12 HV; 19 TOL; 27 STA). - Study 5 from Sagoo P. et al. (J Clin Invest. 2010) published under GEO accession number GSE14655 and comprising two distinct cohorts of patients. The IOT (“Indices of Tolerance”) cohort (5a) corresponds to a European group of patients and totalizes 74 unique samples (8 HV; 10 TOL; 11 MIS; 36 STA; 9 CR). The ITN (“Immune Tolerance in Transplantation”) cohort (5b) corresponds to an American group of patients and totalizes 105 unique samples (20 HV; 22 TOL; 11 MIS; 34 STA; 18 CR). The final data matrix corresponds to the combination of the data from the five independent studies and examines the expression of 1846 meta-genes (MG) across 596 unique blood samples. The 1846 meta-genes correspond to the genes being measured across the five datasets and form a virtual microarray platform (TOL-HUMAN-G1846). The 596 examined samples can be divided into 62 HV, 96 TOL, 32 MIS, 311 STA, 81 CR, and 14 AR.

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Systems biology of gene expression of bladder papilloma cells modulated by a malignant-derived extracellular matrix

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