New Isolation Technique to Study Apoptosis in Human Intestinal Epithelial Cells

Grossmann, Johannes; Maxson, Julie M.; Whitacre, Cecilia M.; Orosz, David E.; Berger, Nathan A.; Fiocchi, Claudio; Levine, Alan D.

doi:10.1016/s0002-9440(10)65545-9

Cited by 105 publications

(66 citation statements)

References 44 publications

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“…More than 97% of the isolated cells were intestinal epithelial cells that were positive for cytokeratin. 38 Studies in our lab using flow cytometry confirmed that less than 3% of contaminating cells stained for the pan-leukocyte marker, CD45 (data not shown).…”

Section: Intestinal Epithelial Cell Isolationmentioning

confidence: 74%

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Epithelial NF-κB Enhances Transmucosal Fluid Movement by Altering Tight Junction Protein Composition after T Cell Activation

Tang¹,

Clayburgh

Mittal³

et al. 2010

The American Journal of Pathology

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In inflammatory bowel disease (IBD) , aberrant activation of innate and adaptive immune responses enhances mucosal permeability through mechanisms not completely understood. To examine the role of epithelial nuclear factor (NF-B) in IBD-induced enhanced permeability , epithelial-specific IB␣ mutant (NF-B super repressor) transgenic (TG) mice were generated. NF-kB activation was inhibited in TG mice, relative to wild-type mice , following T cell-mediated immune cell activation using an anti-CD3 monoclonal antibody. Furthermore , epithelial NF-B super repressor protein inhibited diarrhea and blocked changes in transepithelial resistance and transmucosal flux of alexa350 (0.35 kDa) and dextran3000 (3 kDa). In vivo perfusion loop studies in TG mice revealed reversed net water secretion and reduced lumenal flux of different molecular probes (bovine serum albumin, alexa350, and dextran3000). Cell-imaging and immunoblotting of low-density, detergent-insoluble membrane fractions confirmed that tight junction proteins (occludin, claudin-1 and zona occludens-1) are internalized through an NF-B-dependent pathway. Taken together, these data suggest that IBD-associated diarrhea results from NF-B-mediated tight junction protein internalization and increased paracellular permeability. Thus, reduction of epithelial NF-B activation in IBD may repair defects in epithelial barrier function, reduce diarrhea, and limit protein (eg, serum albumin) losses. Epithelial NF-B activation induced by mucosal T cells, therefore, actively plays a role in opening paracellular spaces to promote transmucosal fluid effux into the intestinal lumen.

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Section: Intestinal Epithelial Cell Isolationmentioning

confidence: 74%

“…38 Lengthwise sections of intestine were washed in 4°C Ca 2ϩ -and Mg ϩ -free Hanks Balanced Salt Solution. The tissue was transferred to CMF-HBSS containing 10 mmol/L dithiothreitol and 50 nmol/L calyculin A (Calbiochem) for 30 minutes, 4°C.…”

Section: Intestinal Epithelial Cell Isolationmentioning

confidence: 99%

Epithelial NF-κB Enhances Transmucosal Fluid Movement by Altering Tight Junction Protein Composition after T Cell Activation

Tang¹,

Clayburgh

Mittal³

et al. 2010

The American Journal of Pathology

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“…We purified intestinal epithelial cells using a modification of a previously published method (80). Briefly, a fresh section of intestine was opened lengthwise and washed in 4°C Ca 2+ -and Mg + -free HBSS (CMF-HBSS).…”

Section: Methodsmentioning

confidence: 99%

Epithelial myosin light chain kinase-dependent barrier dysfunction mediates T cell activation-induced diarrhea in vivo

Clayburgh

Barrett²,

Tang³

et al. 2005

Journal of Clinical Investigation

376

455

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Disruption of the intestinal epithelial barrier occurs in many intestinal diseases, but neither the mechanisms nor the contribution of barrier dysfunction to disease pathogenesis have been defined. We utilized a murine model of T cell-mediated acute diarrhea to investigate the role of the epithelial barrier in diarrheal disease. We show that epithelial barrier dysfunction is required for the development of diarrhea. This diarrhea is characterized by reversal of net water flux, from absorption to secretion; increased leak of serum protein into the intestinal lumen; and altered tight junction structure. Phosphorylation of epithelial myosin II regulatory light chain (MLC), which has been correlated with tight junction regulation in vitro, increased abruptly after T cell activation and coincided with the development of diarrhea. Genetic knockout of long myosin light chain kinase (MLCK) or treatment of wild-type mice with a highly specific peptide MLCK inhibitor prevented epithelial MLC phosphorylation, tight junction disruption, protein leak, and diarrhea following T cell activation. These data show that epithelial MLCK is essential for intestinal barrier dysfunction and that this barrier dysfunction is critical to pathogenesis of diarrheal disease. The data also indicate that inhibition of epithelial MLCK may be an effective non-immunosuppressive therapy for treatment of immune-mediated intestinal disease.

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“…When epithelial cells lose such contacts, a form of apoptosis results, which has been termed anoikis (1). Physiologically, anoikis is important in the regulation of cell number in the intestinal tract, where epithelial cells are shed from villus tips in the small intestine and from the intercrypt epithelium of the colon and die by anoikis (2,3). Pathophysiologically, resistance to anoikis has been associated with metastatic spread of carcinoma cells (4,5), which reflects the ability of malignantly transformed epithelial cells to evade apoptosis when deprived of extracellular matrix attachment during dissemination in lymph or blood.…”

mentioning

confidence: 99%

Antianoikis Effect of Nuclear Factor-κB through Up-regulated Expression of Osteoprotegerin, BCL-2, and IAP-1

Törüner

Fernández-Zapico

Sha

et al. 2006

Journal of Biological Chemistry

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Contact with extracellular matrix provides survival signals to epithelial cells. When epithelial cells lose such contacts, a form of apoptosis results, which has been termed anoikis (1). Physiologically, anoikis is important in the regulation of cell number in the intestinal tract, where epithelial cells are shed from villus tips in the small intestine and from the intercrypt epithelium of the colon and die by anoikis (2, 3). Pathophysiologically, resistance to anoikis has been associated with metastatic spread of carcinoma cells (4, 5), which reflects the ability of malignantly transformed epithelial cells to evade apoptosis when deprived of extracellular matrix attachment during dissemination in lymph or blood. The molecular mechanisms that govern susceptibility to anoikis have not been fully characterized and could provide important insights into epithelial homeostasis and carcinogenesis.It was previously established that the survival of cells cultured without extracellular attachments is promoted by PI 3-K 2 and AKT because inhibition of these kinases increased anoikis (6, 7). Moreover, the anchorage-independent survival of cancer cells that is induced by transforming oncogenes such as Ras and Src depends in part on PI 3-K and AKT (8 -10). However, the molecular mechanisms by which cells activate PI 3-K and AKT after loss of extracellular attachments and through which this pathway blocks anoikis have not been completely characterized; nor have the molecular factors that ultimately control the susceptibility of cells to undergo anoikis been identified.NF-B constitutes a family of transcription factors defined by the presence of a Rel homology domain. When exposed to certain stresses, such as inflammation, infection, oxidative stress, or DNA double strand breaks, NF-B dissociates from inhibitory I B molecules, translocates from cytoplasm to nucleus, and binds to B sequences in the promoter regions of specific target genes, up-regulating their expression (11). Prominent among the more than 150 genes known to be regulated by NF-B proteins are a subset whose products inhibit apoptosis, including BCL-X L , BCL-2, IAP-1 and -2, c-FLICE inhibitory protein, growth arrest and DNA damage associated protein 45␤, and tumor necrosis factor (TNF) receptor-associated proteins 1 and 2 (12). NF-B activation is strongly associated with resistance to apoptosis induced by TNF-␣ (13), ionizing radiation (14), and cancer chemotherapeutic drugs (15). An important role for NF-B in carcinogenesis has been proposed, based on the oncogenicity of v-rel (16), the effect of NF-B in promoting chemotherapy and radiotherapy resistance of cancer cells (17), and the functional requirement for NF-B in tumorigenesis in the colon, breast, and liver (18 -20). Because of the importance of NF-B in apoptosis resistance and carcinogenesis, we suspected that this factor might regulate anoikis.To address this possibility, we evaluated a functional role for NF-B in the regulation of anoikis susceptibility, using a suspension culture model of intestinal e...

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New Isolation Technique to Study Apoptosis in Human Intestinal Epithelial Cells

Abstract: Apoptosis is critically important to development, tissue homeostasis, and the pathogenesis of various diseases.

Cited by 105 publications

References 44 publications

Epithelial NF-κB Enhances Transmucosal Fluid Movement by Altering Tight Junction Protein Composition after T Cell Activation

Epithelial NF-κB Enhances Transmucosal Fluid Movement by Altering Tight Junction Protein Composition after T Cell Activation

Epithelial myosin light chain kinase-dependent barrier dysfunction mediates T cell activation-induced diarrhea in vivo

Antianoikis Effect of Nuclear Factor-κB through Up-regulated Expression of Osteoprotegerin, BCL-2, and IAP-1

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