Cell-Cell Contacts Prevent Anoikis in Primary Human Colonic Epithelial Cells
2006; Elsevier BV; Volume: 132; Issue: 2 Linguagem: Inglês
10.1053/j.gastro.2006.11.017
ISSN1528-0012
AutoresClaudia Hofmann, Florian Obermeier, Monika Artinger, Martin Hausmann, Werner Falk, Juergen Schöelmerich, Gerhard Rogler, Johannes Grossmann,
Tópico(s)Genetic factors in colorectal cancer
ResumoBackground & Aims: Colonic epithelial cells (CECs) receive important survival signals from the extracellular matrix and undergo detachment-induced apoptosis (anoikis) as soon as they lose their cell-matrix anchorage. In contrast to the established role of cell-matrix contact, the role of cell-cell contacts as a physiologic survival factor for CECs is less clear. Methods: Intact CEC crypts gently centrifuged to form a cell aggregate in which cell-cell contacts were maintained. Induction of apoptosis was assessed by Western Blot analysis, colorimetric assays, DNA electrophoresis, 4′,6-diamidino-2-phenylindole staining, and flow cytometry. Activation of survival pathways was analyzed by Western blot. The role of mitogen-activated protein kinase/extracellular signal–regulated kinase (MEK)/extracellular signal–regulated kinase (Erk)1/2, epidermal growth factor receptor, phosphatidylinositol 3-kinase (PI3-K), and Src signaling was investigated using specific inhibitors. Results: Despite a complete loss of cell-matrix adhesion after CEC isolation, activation of caspases was blocked and anoikis was prevented when cell-cell contacts were preserved. CECs with preserved cell-cell contacts exhibited a rapid dephosphorylation of focal adhesion kinase. Aggregated CECs had stable levels of active β-catenin and phosphorylated Akt, Erk1/2, and epidermal growth factor receptor, but CECs undergoing anoikis rapidly degraded β-catenin and dephosphorylated Akt. Inhibition of Src- and PI3-K–dependent signaling reversed the antiapoptotic effect of cell-cell contact preservation, while inhibition of the MEK pathway had no effect. Conclusions: Integrity of cell-cell contacts compensates for the loss of cell-matrix contact-mediated survival signals in CECs and prevents apoptosis. Cell-cell contact-triggered CEC survival involves antiapoptotic signaling through β-catenin-, Src-, and PI3-K/Akt- but not through MEK- and focal adhesion kinase–dependent pathways. Background & Aims: Colonic epithelial cells (CECs) receive important survival signals from the extracellular matrix and undergo detachment-induced apoptosis (anoikis) as soon as they lose their cell-matrix anchorage. In contrast to the established role of cell-matrix contact, the role of cell-cell contacts as a physiologic survival factor for CECs is less clear. Methods: Intact CEC crypts gently centrifuged to form a cell aggregate in which cell-cell contacts were maintained. Induction of apoptosis was assessed by Western Blot analysis, colorimetric assays, DNA electrophoresis, 4′,6-diamidino-2-phenylindole staining, and flow cytometry. Activation of survival pathways was analyzed by Western blot. The role of mitogen-activated protein kinase/extracellular signal–regulated kinase (MEK)/extracellular signal–regulated kinase (Erk)1/2, epidermal growth factor receptor, phosphatidylinositol 3-kinase (PI3-K), and Src signaling was investigated using specific inhibitors. Results: Despite a complete loss of cell-matrix adhesion after CEC isolation, activation of caspases was blocked and anoikis was prevented when cell-cell contacts were preserved. CECs with preserved cell-cell contacts exhibited a rapid dephosphorylation of focal adhesion kinase. Aggregated CECs had stable levels of active β-catenin and phosphorylated Akt, Erk1/2, and epidermal growth factor receptor, but CECs undergoing anoikis rapidly degraded β-catenin and dephosphorylated Akt. Inhibition of Src- and PI3-K–dependent signaling reversed the antiapoptotic effect of cell-cell contact preservation, while inhibition of the MEK pathway had no effect. Conclusions: Integrity of cell-cell contacts compensates for the loss of cell-matrix contact-mediated survival signals in CECs and prevents apoptosis. Cell-cell contact-triggered CEC survival involves antiapoptotic signaling through β-catenin-, Src-, and PI3-K/Akt- but not through MEK- and focal adhesion kinase–dependent pathways. 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Progress on isolation and short-term ex-vivo culture of highly purified non-apoptotic human intestinal epithelial cells (IEC).Eur J Cell Biol. 2003; 82: 262-270Crossref PubMed Scopus (49) Google Scholar Briefly, normal human colonic mucosa from surgical specimens obtained from patients undergoing surgery for large bowel neoplasia (>10 cm from the tumor) were cut into small strips. Mucus was removed by incubation for 30 minutes at room temperature in 1 mmol/L dithiothreitol (Sigma, Taufkirchen, Germany) in 50 mL Hank's balanced salt solution (PAA, Linz, Austria). Mucosal strips were incubated in 1 mmol/L EDTA (Sigma) for 10 minutes at 37°C, briefly rinsed in Hank's balanced salt solution, and transferred to tubes containing fresh Hank's balanced salt solution at room temperature. Tubes were shaken vigorously 5–10 times. Mucosal strips were removed by passing the slurry over a coarse mesh (400 μm; Carl Roth GmbH, Karlsruhe, Germany). The suspension containing the detached CEC crypts was passed over the mesh filter (80-μm pore size; Sefar, Kansas City, MO), and intact CEC crypts were eluted by inverting the filter in serum-free culture medium (keratinocyte serum-free medium; Gibco-BRL, Eggenstein, Germany). Using this method, CECs were purified as intact crypts; it is important to note that cell-cell contacts within the CEC crypts were preserved, whereas cell-matrix contact was lost. To determine the role of cell-cell anchorage, freshly isolated CEC crypts were gently spun to form a cell aggregate (pellet) before disruption of CEC cell-cell contacts (Figure 1). Each pellet was generated from 3- to 5-mL aliquots of the suspension by centrifugation at 100g and 4°C for 5 minutes, in a manner similar to the method of Maldonado et al.43Maldonado R.A. Irvine D.J. Schreiber R. Glimcher L.H. A role for the immunological synapse in lineage commitment of CD4 lymphocytes.Nature. 2004; 431: 527-532Crossref PubMed Scopus (162) Google Scholar As control, CECs were liberated from isolated crypts by suspension in a polypropylene tube on a whip-shaker, inducing apoptosis as described previously.44Grossmann J. Mohr S. Lapentina E.G. Fiocchi C. Levine A.D. Sequential and rapid activation of select caspases during apoptosis of normal intestinal epithelial cells.Am J Physiol. 1998; 274: G1117-G1124PubMed Google Scholar Pellets and CECs in suspension were incubated at 37°C. At the indicated time points, cells were harvested by centrifugation at 4°C and analyzed further. Cells were fixed in 0.1 mol/L cacodylate-buffered Karnovsky solution (2.5% glutaraldehyde and 1% paraformaldehyde) overnight at room temperature, postfixed in 1% osmium tetroxide (2 hours) at pH 7.3, dehydrated in graded ethanols, and embedded in the EmBed-812 epoxy resin (all reagents from Science Services, Munich, Germany). After 48-hour heat polymerization at 60°C, semithin (0.8-μm) sections were cut and stained with toluidine blue; after selection of appropriate areas of interest, the Epon block was trimmed for ultrathin sectioning. Ultrathin (80-nm) sections were cut with a diamond knife on a Reichert Ultracut-S ultramicrotome (Reichert, Munich, Germany). Double contrast was achieved by incubation in aqueous 2% uranyl acetate and lead citrate solutions for 10 minutes each. The sections were examined in an LEO912AB electron microscope (Carl Zeiss, Jena, Germany) operating at 80 kV. CECs in suspension or in pellet were harvested at the indicated time points. DNA was extracted as described45Sambrook J. Fritsch E.F. Maniatis T. Commonly used techniques in molecular cloning.molecular cloning. 3rd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar and separated by 1.5% agarose gel electrophoresis. Gels were stained with ethidium bromide and visualized with UV light. Cells in suspension and in pellet, respectively, were harvested at the indicated time points and centrifuged at 100g and 4°C for 5 minutes. Cells were resuspended in 70% ice-cold methanol and stored at −20°C until analysis, typically within 24 hours. Flow cytometry analysis was performed using propidium iodine incorporation as described.26Grossmann J. Walther K. Artinger M. Kiessling S. Steinkamp M. Schmautz W.K. Stadler F. Bataille F. Schultz M. Scholmerich J. Rogler G. Progress on isolation and short-term ex-vivo culture of highly purified non-apoptotic human intestinal epithelial cells (IEC).Eur J Cell Biol. 2003; 82: 262-270Crossref PubMed Scopus (49) Google Scholar A minimum of 5 × 103 cells was examined per sample. Gating was performed in the side scatter plot to exclude debris and on the pulse width plot to exclude remaining cell aggregates from analysis. CECs were fixed on microscope slides by cytospin centrifugation, stained with 4′,6-diamidino-2-phenylindole–containing Vectashield mounting medium (Vector, Burlingame, CA), and analyzed by fluorescence microscopy. CEC cytosol was extracted as described46Grossmann J. Artinger M. Grasso A.W. Kung H.J. Scholmerich J. Fiocchi C. Levine A.D. Hierarchical cleavage of focal adhesion kinase by caspases alters signal transduction during apoptosis of intestinal epithelial cells.Gastroenterology. 2001; 120: 79-88Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar at the indicated times after incubation. Proteins were separated by sodium dodecyl sulfate/polyacrylamide electrophoresis, transferred to membranes, and probed with antibodies that were diluted as follows: anti–caspase-2 (1:2000; Alexis Biochemicals, San Diego, CA); anti–caspase-3 (1:3000; Transduction Laboratories, Lexington, KY); anti–caspase-7 (1:3000; Transduction Laboratories); anti–caspase-8 (1:3000; BioCheck, Münster, Germany); anti–caspase-9 (1:1000; Biomol, Hamburg, Germany); anti–focal adhesion kinase (FAK) (1:666; Chemicon, Temecula, CA); anti-pY397 FAK (1:1000; Biosource, Camarillo, CA); anti-Akt (1:1000) and anti-pAkt (Thr308 and Ser473) (1:1000; Cell Signaling, Beverly, MA); anti-pEGFR, anti–epidermal growth factor receptor (EGFR), anti-pErk1/2, anti–extracellular signal–regulated kinase (Erk) 1/2, anti-pSrc (Y416), and anti-Src (all 1:1000; Cell Signaling); and anti–β-catenin (1:3000; Biomol) and anti–active-β-catenin (1:2000; Biomol). Equal loading of the cytosolic samples was demonstrated by reprobing membranes with an anti–β-actin antibody (1:10,000; Chemicon, Hofheim, Germany). Caspase-3–like activity in CEC cytosolic extracts was determined using the synthetic substrate N-acetyl-Asp-Glu-Val-Asp-para-nitroanilide (Ac-DEVD-pNA; Biomol, Plymouth Meeting, PA). Extracts (30 μg protein) were incubated with Ac-DEVD-pNA (200 μmol/L) for 2 hours at 30°C. Caspase-like activity was quantified by absorption at 405 nm. As a positive control for caspase-3–like activity, extracts from CECs undergoing anoikis for 2 hours were used. Bovine serum albumin solution served as a negative control. To block relevant signaling pathways in CECs, freshly isolated crypts were preincubated with specific inhibitors for 10 minutes at room temperature, followed by centrifugation and pellet incubation for 2 hours at 37°C. As a positive control for the induction of apoptosis, CECs were left untreated and incubated without aggregation. The following inhibitors were used: LY294002 (PI3-K inhibitor; Cell Signaling), U0126 (mitogen-activated protein kinase/extracellular signal–regulated kinase [MEK] inhibitor; Cell Signaling), tyrphostin AG1478 (EGFR inhibitor; Calbiochem, Darmstadt, Germany), and PP1 (Src inhibitor; Biomol, Hamburg, Germany). Freshly isolated CEC crypts were incubated in culture medium containing a functional E-cadherin ligand (dimeric E-cadherin chimera protein, ie, human E-cadherin ectodomain fused to the Fc fragment of human immunoglobulin G1; 10 μg/mL; R&D Systems, Minneapolis, MN). E-cadherin-Fc was preincubated with CEC crypts for 15 minutes at 4°C. Anti-human immunoglobulin G specific for the Fc fragment (6 μg/mL; Jackson ImmunoResearch, Cambridgeshire, England) was added to trigger E-cadherin-Fc clustering at the cell membrane, and CECs were transferred to 37°C on a shaker for 1 hour. Data are expressed as mean ± SD. Statistical analysis was performed using the Student t test. Differences were considered significant with a P value of <.05. To assess the role of cell-cell contacts in preventing CEC apoptosis, it was necessary to establish a model for the maintenance of CEC cell-cell contacts ex vivo. To preserve existing cell-cell contacts, freshly isolated crypts were gently centrifuged to form a cell aggregate (pellet; P-CECs) before disaggregation of cell-cell contacts in the CEC crypts could occur. As control, CECs were kept in suspension (S-CECs) to induce loss of cell-cell contact and consecutively anoikis (Figure 1). First, we wanted to demonstrate that CEC cell-cell contacts can be preserved by aggregate formation and to investigate how aggregate formation influences CEC morphology. For this reason, electron microscopy was performed (Figure 2). Immediately after isolation, CEC crypts displayed stable apical cell-cell contacts. Numerous microvilli were detectable (Figure 2A). Although pellet incubation slightly deformed apical surfaces of P-CECs (due to the physical strain during centrifugation), sites of intercellular adhesion remained intact (Figure 2B). Apoptosis and necrosis were not detected. In contrast, S-CECs showed an apoptotic phenotype, displaying strong chromatin condensation and formation of apoptotic bodies. Cells were shrunken, and crypt structure was completely disintegrated (Figure 2C). These data show that CEC aggregation is capable of preserving cell-cell contacts, cell viability, and cellular morphology. To investigate the effect of maintaining cell-cell contacts on the apoptotic machinery, the activation of distinct caspases was examined by Western blot analysis (Figure 3, Figure 4). First, the activation of initiator caspases was assessed. In S-CECs, caspase-2 (p48) was cleaved to form the p33 fragment after 30 minutes. The amount of activated caspase-2 rapidly increased during the incubation, and after 120 minutes, caspase-2 was detected exclusively in its processed form (Figure 3A). A similar activation pattern could be observed for caspase-9 (Figure 3B); the appearance of the p34 fragment was observed after 30 minutes in S-CECs, and the levels of processed caspase-9 strongly increased thereafter. In contrast, P-CECs showed less activation of caspase-2 and caspase-9 (Figure 3A and B). Caspase-8 remained inactive initially in S-CECs (p48 and p45 isoforms were present, exclusively). However, 30–60 minutes after detachment, the prodomain was processed, forming p43 and p41 fragments; the p18 subunit of activated caspase-8 became detectable after 90 minutes (Figure 3C). In P-CECs, the amount of pro–caspase-8 remained constant for 120 minutes, and only slight processing of the prodomain to p43/p41 fragments could be observed. In contrast to S-CECs, no active form of caspase-8 (p18) was detectable at any time (Figure 3C).Figure 4Maintenance of cell-cell contacts prevents effector caspase activation. Cytosolic extracts from S-CECs and P-CECs were prepared at the indicated times and analyzed by Western blot for the a
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