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 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 37º 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/250µl. 250µl 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
