Comparison of parental vs tumor-derived imortalized mouse kidney epithelial cell (iBMK) lines
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE10682 | GEO | 2008/03/25
SECONDARY ACCESSION(S): PRJNA107669
REPOSITORIES: GEO
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Project description:Most tumors are epithelial-derived, and although disruption of polarity and aberrant cellular junction formation is a poor prognosticator in human cancer, the role of polarity determinants in oncogenesis is poorly understood. Using in vivo selection, we identified a mammalian orthologue of the Drosophila polarity regulator crumbs as a gene whose loss of expression promotes tumor progression. Immortal baby mouse kidney epithelial (iBMK) cells selected in vivo to acquire tumorigenicity displayed dramatic repression of crumbs3 (crb3) expression associated with disruption of tight junction formation, apicobasal polarity, and contact-inhibited growth. Restoration of crb3 expression restored junctions, polarity and contact inhibition, while suppressing migration and metastasis. These findings suggest a role for mammalian polarity determinants in suppressing tumorigenesis that may be analogous to the well-studied polarity tumor suppressor mechanisms in Drosophila. Experiment Overall Design: Gene Expression analysis was carried out using the Affymetrix Mouse 430 A 2.0 chips representing about 22,629 full-length genes and ESTs. Initial scaling was done using the Affymetrix Microarray Suite Expression Software version 5.0 and subsequent analysis was done using GeneSpring Software version 6.1 ( Silicon Genetics). (Tumor derived cell lines) /in vivo selected cell lines were compared to the parental cell lines from which they were derived. We had two parental cell line samples which were defined as replicates and treated as a single sample and 4 cell lines namely TD A, B, C and D derived from the parents. Three different filtering conditions were applied. First, we applied Expression Percentage Restriction retaining only those genes which had a raw expression value of 80.0 or more in at least 1 out of the 5 samples being compared. This enabled us to filter out the low-intensity values while retaining genes that may have a very low expression level in one sample but may be switched on in another sample. The cut-off value of 80.00 was determined by analyzing the raw data of each of the chips being compared and estimating the average background expression value. Of the 22,629 genes represented on the chips, 11,418 genes were retained. Secondly, we filtered on data quality selecting for genes with a Flag value of Present (P) in all the samples being compared. 10,126 genes were retained after applying this filter. We then carried out a pair-wise fold-change analysis comparing each tumor derived cell line to the parental cell-line. We retained genes that underwent at least 2 folds or more up-regulation or down-regulation in the tumor derived cell l ines, compared to the parentals. 1,010 genes were retained after this step. In order to identify genes that underwent statistically significant changes in gene expression in the tumor derived cell-lines when compared to the parentals we applied a statistical filter analysis and performed the Studentâ??s-t-test with a p value cut-off of 0.05. Multiple testing correction was turned off. We found 123 genes that were over-expressed and 5 that were underexpressed. We carried out two-way hierarchical clustering using the GeneSpring Software 6.1 using standard correlation as similarity metric. We also analyzed our data using Venn Diagrams in order to identify the statistically significant genes that were common to all 4 tumor derived cell-lines. We obtained 55 that were up-regulated while there were 5 which were down-regulated and common to all four cell-lines.
2008-04-04 | E-GEOD-10682 | biostudies-arrayexpress
Project description:Hepatocytes are polarized epithelial cells whose function depends upon their ability to distinguish between the apical and basolateral surfaces that are located at intercellular tight junctions. It has been proposed that the signaling cascades that originate at these junctions influence cellular activity by controlling gene expression in the cell nucleus. To assess the validity of this proposal with regard to hepatocytes, we depleted expression of the tight junction protein junctional adhesion molecule-A (JAM-A) in the HepG2 human hepatocellular carcinoma cell line. Reduction of JAM-A resulted in a striking change in cell morphology, with cells forming single-layered sheets instead of the normal multi-layered clusters. In the absence of JAM-A, other tight junction proteins were mislocalized, and canaliculi, which form the apical face of the hepatocyte, were consequently absent. While most changes in gene expression were modest, there was a strong transcriptional induction of the adherens junction protein E-cadherin in cells with reduced levels of JAM-A. This increase in E-cadherin was partially responsible for the observed alterations in cell morphology and mislocalization of tight junction proteins. We therefore propose that we have uncovered a novel mechanism for crosstalk between specific components of tight and adherens junctions that can be utilized to regulate adhesion between hepatic cells and to maintain hepatocyte cell polarity. Experiment Overall Design: HepG2 cells were transduced with lentiviruses and stably selected using puromycin. Comparison of four controls and five JAM-A knockdown samples.
2008-06-14 | E-GEOD-6080 | biostudies-arrayexpress
Project description:Hepatocytes are polarized epithelial cells whose function depends upon their ability to distinguish between the apical and basolateral surfaces that are located at intercellular tight junctions. It has been proposed that the signaling cascades that originate at these junctions influence cellular activity by controlling gene expression in the cell nucleus. To assess the validity of this proposal with regard to hepatocytes, we depleted expression of the tight junction protein junctional adhesion molecule-A (JAM-A) in the HepG2 human hepatocellular carcinoma cell line. Reduction of JAM-A resulted in a striking change in cell morphology, with cells forming single-layered sheets instead of the normal multi-layered clusters. In the absence of JAM-A, other tight junction proteins were mislocalized, and canaliculi, which form the apical face of the hepatocyte, were consequently absent. While most changes in gene expression were modest, there was a strong transcriptional induction of the adherens junction protein E-cadherin in cells with reduced levels of JAM-A. This increase in E-cadherin was partially responsible for the observed alterations in cell morphology and mislocalization of tight junction proteins. We therefore propose that we have uncovered a novel mechanism for crosstalk between specific components of tight and adherens junctions that can be utilized to regulate adhesion between hepatic cells and to maintain hepatocyte cell polarity. Keywords: HepG2 cells
2007-07-01 | GSE6080 | GEO
Project description:In a genome-wide screen of human cancer cell lines, we identified homozygous intragenic microdeletions involving genes encoding components of the apical-basal cell polarity complexes. Among these, PARD3 is disrupted in cell lines and primary tumors from squamous carcinomas and glioblastomas. Reconstituting PARD3 expression in both cell types restores tight junctions and retards contact-dependent proliferation. Searching specifically for small intragenic microdeletions using high-resolution genomic arrays may be complementary to other genomic deletion screens and resequencing efforts in identifying new tumor suppressor genes. 444 human cancer cell lines of diverse tissue origin and 6 EBV-transformed B-lymphocyte cell lines (Coriell Institute for Medical Research, Camden, NJ) as controls.
2011-11-03 | E-GEOD-20306 | biostudies-arrayexpress
Project description:In a genome-wide screen of human cancer cell lines, we identified homozygous intragenic microdeletions involving genes encoding components of the apical-basal cell polarity complexes. Among these, PARD3 is disrupted in cell lines and primary tumors from squamous carcinomas and glioblastomas. Reconstituting PARD3 expression in both cell types restores tight junctions and retards contact-dependent proliferation. Searching specifically for small intragenic microdeletions using high-resolution genomic arrays may be complementary to other genomic deletion screens and resequencing efforts in identifying new tumor suppressor genes.
2011-11-03 | GSE20306 | GEO
Project description:The blood-brain barrier (BBB) is an evolutionary conserved tissue interface that possesses potent chemical protection properties functioning to strictly modulate the central nervous system (CNS) microenvironment. These same properties, including tight cellular junctions and efflux transporters, also limit access of CNS-active pharmaceuticals. For this reason, understanding the molecular mechanisms that regulate BBB chemical protection is of great biomedical interest. The BBB of Drosophila consists of two surface glia layers that completely surround the brain. This tissue interface contains both “tight” cellular junctions (termed septate junctions) and drug efflux transporters; thus, the Drosophila BBB can potentially serve as a model for understanding complex regulation of BBB physiology. In this study, we show reciprocal compensatory responses following disruption of critical BBB genes: deletion of the septate junction regulator Moody causes increased drug efflux and up-regulation of the P-glycoprotein ortholog Mdr65; conversely, disruption of Mdr65 expression causes increased septate junction tightness and up-regulation of Moody. We reveal these homeostatic interactions with physiologic observations, gene expression data, and anatomical images of the BBB surface. Whole brain microarray data point to responses that are consistent with our physiologic observations and these responses are likely localized to the BBB. To our knowledge, this is the first observation of a reciprocal compensatory interaction at a tissue barrier. Furthermore, this study paves the way for future studies elucidating the direct pathways that link GPCR signaling and drug transporter regulation at the BBB. The main comparisons are between WT_new, C17 (Moody null), and PMdr65 (Mdr65 null). Since WT_new were processed on a different day than C17 and PMdr65, we also included another WT sample (WT_old) to control for genes that change depending on batch effects. Since the mutants are also in a different genetic background than WT, we also included a control that is in a similar background (UAS_control).
2013-12-31 | E-GEOD-46550 | biostudies-arrayexpress
Project description:The epithelial cell layer that lines the fish gill controls the paracellular permeation of chemicals through tight junctions. The integrity of tight junctions can be affected by inflammation, which is likely to impact the branchial bioavailability of chemicals. In this study, we experimentally induced inflammation in the rainbow trout gill cell line RTgill-W1 via exposure to bacterial lipopolysaccharides (LPS). We then co-exposed the cells to extracts of oil sands process-affected water (OSPW), which contain a complex mixture of toxicologically relevant chemicals. Cells exposed to LPS showed a significant reduction in transepithelial electrical resistance (TEER), an indicator of tight junction integrity, after 24 h of exposure. Quantitative RT-PCR analysis determined that the abundance of transcripts of genes coding for tight junction proteins (Claudin 28b and 10e) was significantly decreased in cells exposed to 20, 50, and 100 mg L-1 LPS. Chemical analysis revealed a significant increase in permeation of constituents of OSPW across the gill cell epithelial layer at all studied LPS concentrations. These in vitro findings were confirmed in vivo in rainbow trout fingerlings exposed to both LPS and 10% OSPW for 48 h, which similarly resulted in an increase in chemical uptake relative to fish exposed to OSPW alone. This research demonstrated that inflammation of gill epithelia and the resulting disruption of tight junction integrity could lead to significantly greater uptake of potentially harmful chemicals from the environment, which has important implications for risk assessment.
2022-02-28 | GSE182953 | GEO
Project description:Our data demonstrate that overexpression of the polarity protein Crb3 elicits changes in MCF-10A cells that culminate in an increase in the release of amphiregulin (AR) and the subsequent activation of EGFR signaling to drive proliferation. Microarray analysis was performed to define global changes in the transcriptional landscape induced by Crb3. Results provide insight into a FERM domain protein (EBP41L4B) required for Crb3 mediated induction of proliferation.
2018-12-26 | GSE76610 | GEO
Project description:To study the role of hepatic nuclear factor _ alpha (HNF4a_ in hepatogenesis, we used loxP-Cre technology to eliminate it from developing mouse livers. A comparison of control and experimental livers revealed that hepatocytes lacking HNF4a_failed to fully differentiate. Specifically, HNF4a null liver cells failed to express genes that are markers of mature hepatocytes including Gys2, Pck1, and G6pc. These cells also failed to form normal cellular junctions, which are critical for establishment of the hepatic epithelium [Parviz et al. (2003) Nat.Genet. 34, 292-96]. Because HNF4a functions as a transcription factor, we hypothesized that it regulates liver development by controlling the expression of genes whose products are essential in executing cellular differentiation and morphogenesis. To identify these genes, we compared expression profiles between HNF4a null and wild type E18.5 fetal livers using Affymetrix gene arrays. We identified 564 genes whose expression was decreased by at least 2.5-fold and 34 genes whose expression was increased by at least 2.5-fold when HNF4a was eliminated from hepatocytes. These represent genes involved in diverse molecular pathways, reflecting the pleiotropic phenotype of HNF4a null livers. Our goal was to use the information from the gene arrays to define the mechanisms through which HNF4a controls the generation of the hepatic epithelium. We identified 27 genes from our list of downregulated genes with either defined or predicted roles in cellular junctions and/or adhesion. Most striking was that loss of HNF4a altered the expression of genes encoding proteins involved in virtually all types of cellular junctions including tight junctions (Cldn1, Cldn-2, Cldn-12, Ocln, F11r/Jam1, Cxadr, Crb3), adherens junctions (Cdh1), desmosomes (Dsc2, Pkp2, Krt2-8), and gap junctions (Gjb1, Gjb2). Using immunoblotting and immunohistochemistry, we found that the expression of proteins representing each of these junction types was reduced or absent. Analysis of these genes for the presence of putative HNF4a binding sites revealed that 25 of 27 contain such sites. We have used chromatin immunoprecipitation to confirm that HNF4a occupies several of these sites in vivo, including Cdh1, Cldn1, and F11r/Jam1. From these data, we conclude that HNF4_ is a central mediator of the fully differentiated hepatocyte gene expression program and that it orchestrates the expression of cell junction and adhesion proteins required for establishing the hepatic epithelium. Experiment Overall Design: comparison of three hnf4 null livers to three control.
2007-12-01 | E-GEOD-3126 | biostudies-arrayexpress
Project description:Tight junction (TJ) proteins (Tjps), Tjp1 and Tjp2, are tight junction-associated scaffold proteins that bind to the transmembrane proteins of tight junctions and the underlying cytoskeleton. In this study, we first analyzed the tumorigenic characteristics of B16-F10 melanoma cells, including cell proliferation, migration, invasion, metastatic potential, and the expression patterns of related proteins, after the CRISPR-Cas9-mediated knockout (KO) of Tjp genes. The proliferation of Tjp1 and Tjp2 KO cells significantly increased in vitro. Other tumorigenic characteristics, including migration and invasion, were significantly enhanced in Tjp1 and Tjp2 KO cells.
2024-08-01 | GSE240070 | GEO