Project description:CELL CULTURE: The human intestinal epithelial cell line Caco-2 (obtained from Dr. Stanley Falkow, Stanford University) was used to study the genome-wide expression program underlying the transition from cell proliferation to establishment of a polarized epithelial layer. Cells were first grown in sparse culture on plastic p15 dishes to generate a population of "contact naive" cells. These cells were combined and plated at confluency on Costar filter inserts. The day cells were plated on filters is designated the zero time-point. Cells were cultured for 26 days, during which time they develop structural and functional polarity. Media was changed every other day. Triplicate samples were harvested at eleven time-points over the time course for microarray analysis. The entire time course was performed twice. Three control arrays are included in this data set; (1) to determine how the gene-expression patterns in Caco-2 cells grown on a plastic dish compares to the expression profiles identified in Caco-2 cells grown on Costar filter inserts (microarray shcu182, data not shown), (2) to tested whether feeding the cells at different time-points had an effect on the Caco-2 gene-expression program. All samples taken (except at the day zero time-point) were fed with fresh media one day prior to harvesting the sample. During TC3 and TC4, we performed two test arrays analyzing the transcriptional program in Caco-2 cells that were fed with fresh cell culture media 4h prior to taking the 7D time-point (microarrays shcu177 and shcu178, indicated with * in table S1). Groups of assays that are related as part of a time series. See additional information on overall design in Series supplementary file. ABSTRACT: Although there is considerable evidence implicating post-translational mechanisms in the development of epithelial cell polarity, little is known about the patterns of gene expression and transcriptional regulation during this process. We characterized the temporal program of gene expression during cell-cell adhesion-initiated polarization of human Caco-2 cells in tissue culture, which develop structural and functional polarity similar to that of enterocytes in vivo. A distinctive switch in gene expression patterns occurred upon formation of cell-cell contacts between neighboring cells. Expression of a cluster of genes involved in cell proliferation switched off and concomitantly the expression of genes related to functional specializations of polarized epithelial cells was induced. Induction of these latter genes correlated with formation of important structural and functional features related to enterocyte differentiation and establishment of structural and functional cell polarity. Coordinated expression of genes encoding components of functional cell structures were often observed, indicating temporal control of expression and assembly of multiprotein complexes. Changes in gene expression patterns during Caco-2 cell differentiation in vitro paralleled those in terminally differentiating enterocytes migrating up the intestinal crypt-villus axis in vivo, despite the lack of stromal cells and gradients of morphogens in this tissue culture model. Keywords: time_series_design Computed

Project description:In response to polarization cues, cultured Caco-2 cells, a human colon adenocarcinoma-derived cell line, form a polarized epithelium resembling normal enterocytes. We investigated potential signaling mechanisms activated by Caco-2 cells that might trigger the genome-wide transcriptional reprogramming that accompanies polarization (Saaf et al, submitted-I). cDNA microarrays were used to compare the transcriptional profile of Caco-2 polarization to the gene expression profiles of normal human colon and colon tumors. The transcript profile of proliferating, non-polarized Caco-2 cells has striking parallels to the gene expression profile of human colon cancer in vivo. However, as Caco-2 cells develop polarity, the gene expression profile shifts to one more closely resembling that of normal colon tissue, suggesting that the underlying regulatory mechanisms that mediate Caco-2 cell polarization are similar to those that occur during in vivo enterocyte differentiation. We show that transcriptional re-programming of Caco-2 cells during development of cell polarity occurs in the context of signaling pathways that are regulated in a manner that is remarkably similar to those in normal intestinal development. For example, transcriptional targets of the Wnt pathway are tightly regulated during Caco-2 cell polarization, mimicking the gradient of Wnt-mediated transcription in the crypt (high expression) to villus (low expression) axis in human intestine. However, Caco-2 cells lack full-length APC necessary for normal Wnt-regulated degradation of beta-catenin. Biochemical analysis indicates that regulation of the Wnt pathway occurs in the nucleus at the level of activation of target genes by the beta-catenin-TCF complex, revealing a novel additional mechanism by which intestinal cells may regulate Wnt signaling during their maturation. In addition, other signaling pathways including Notch, BMP, Hedgehog, and growth factor, were temporally regulated during Caco-2 cell polarization. Surprisingly, modulation of these signaling pathways in Caco-2 cells occurs in the absence of morphogen gradients and interactions with stromal cells characteristic of enterocyte differentiation in situ. This dataset contains gene expression profiles of 9 normal colon samples and 15 colon tumor samples. Samples of tumor and normal colon mucosa were collected from colon cancer resection from Department of Surgery, Queen Mary Hospital University of Hong Kong. Tissue was frozen in liquid nitrogen within 30 min of resection. Nonneoplastic mucosa from colon was dissected free of muscle and histologically confirmed to be tumor free by frozen section. Total RNA was extracted using Trizol (Invitrogen, Carlsbad, CA) from each tissue sample and processed for microarray hybridization. A disease state experiment design type is where the state of some disease such as infection, pathology, syndrome, etc is studied. Disease State: Tumor/Normal colon samples Using regression correlation

Project description:CELL CULTURE: The human intestinal epithelial cell line Caco-2 (obtained from Dr. Stanley Falkow, Stanford University) was used to study the genome-wide expression program underlying the transition from cell proliferation to establishment of a polarized epithelial layer. Cells were first grown in sparse culture on plastic p15 dishes to generate a population of "contact naive" cells. These cells were combined and plated at confluency on Costar filter inserts. The day cells were plated on filters is designated the zero time-point. Cells were cultured for 26 days, during which time they develop structural and functional polarity. Media was changed every other day. Triplicate samples were harvested at eleven time-points over the time course for microarray analysis. The entire time course was performed twice. Three control arrays are included in this data set; (1) to determine how the gene-expression patterns in Caco-2 cells grown on a plastic dish compares to the expression profiles identified in Caco-2 cells grown on Costar filter inserts (microarray shcu182, data not shown), (2) to tested whether feeding the cells at different time-points had an effect on the Caco-2 gene-expression program. All samples taken (except at the day zero time-point) were fed with fresh media one day prior to harvesting the sample. During TC3 and TC4, we performed two test arrays analyzing the transcriptional program in Caco-2 cells that were fed with fresh cell culture media 4h prior to taking the 7D time-point (microarrays shcu177 and shcu178, indicated with * in table S1). Groups of assays that are related as part of a time series. See additional information on overall design in Series supplementary file. ABSTRACT: Although there is considerable evidence implicating post-translational mechanisms in the development of epithelial cell polarity, little is known about the patterns of gene expression and transcriptional regulation during this process. We characterized the temporal program of gene expression during cell-cell adhesion-initiated polarization of human Caco-2 cells in tissue culture, which develop structural and functional polarity similar to that of enterocytes in vivo. A distinctive switch in gene expression patterns occurred upon formation of cell-cell contacts between neighboring cells. Expression of a cluster of genes involved in cell proliferation switched off and concomitantly the expression of genes related to functional specializations of polarized epithelial cells was induced. Induction of these latter genes correlated with formation of important structural and functional features related to enterocyte differentiation and establishment of structural and functional cell polarity. Coordinated expression of genes encoding components of functional cell structures were often observed, indicating temporal control of expression and assembly of multiprotein complexes. Changes in gene expression patterns during Caco-2 cell differentiation in vitro paralleled those in terminally differentiating enterocytes migrating up the intestinal crypt-villus axis in vivo, despite the lack of stromal cells and gradients of morphogens in this tissue culture model. Keywords: time_series_design

Dataset Information

Escherichia coli alpha-hemolysin HlyA induces host cell polarity changes, epithelial barrier dysfunction and cell detachment in human colon carcinoma Caco-2 cell model via PTEN-dependent dysregulation of cell junctions

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure