Volume 132, Issue 2, Pages 587-600 (February 2007) Cell-Cell Contacts Prevent Anoikis in Primary Human Colonic Epithelial Cells  Claudia Hofmann, Florian Obermeier, Monika Artinger, Martin Hausmann, Werner Falk, Juergen Schoelmerich, Gerhard Rogler, Johannes Grossmann  Gastroenterology  Volume 132, Issue 2, Pages 587-600 (February 2007) DOI: 10.1053/j.gastro.2006.11.017 Copyright © 2007 AGA Institute Terms and Conditions

Figure 1 Experimental approach to investigate the role of cell-cell contacts in CEC anoikis. Isolated CECs were gently spun to form a cell aggregate (pellet), preserving CEC cell-cell contacts. Loss of cell-cell contacts was induced by keeping the cells in suspension. Gastroenterology 2007 132, 587-600DOI: (10.1053/j.gastro.2006.11.017) Copyright © 2007 AGA Institute Terms and Conditions

Figure 2 Maintenance of cell morphology within P-CECs. CECs were isolated and either (A) directly fixed or incubated for 2 hours (B) in a pellet or (C) in suspension. Fixed preparations were examined for cell-cell contacts by transmission electron microscopy (original magnification: left panels, 5000×; right panels, 20,000×). Squares indicate the areas magnified in the right panels. After 2 hours of incubation, P-CECs displayed intact cell junctions and no signs of apoptosis, whereas S-CECs showed an apoptotic phenotype and a completely disaggregated crypt structure. Gastroenterology 2007 132, 587-600DOI: (10.1053/j.gastro.2006.11.017) Copyright © 2007 AGA Institute Terms and Conditions

Figure 3 Prevention of initiator caspase activation by maintenance of cell-cell contacts. Cytosolic extracts from S-CECs and P-CECs were prepared at the indicated times and analyzed by Western blot for the activation of (A) caspase-2, (B) caspase-9, and (C) caspase-8. Caspase-2 (p48) and caspase-9 (p46) were activated almost instantly in S-CECs, as demonstrated by removal of their prodomains to yield a p33 and p34 fragment, respectively. Caspase-8 (p48 and p45 isoforms) remained initially inactive in S-CECs, but its prodomain was removed to yield p43 and p41 fragments 60 minutes after detachment. The p18 subunit of caspase-8 was detected after 90–120 minutes in S-CEC preparations. In P-CECs, there was reduced activation of initiator caspase-2, caspase-9, and caspase-8 (data representative of 4 experiments). Gastroenterology 2007 132, 587-600DOI: (10.1053/j.gastro.2006.11.017) Copyright © 2007 AGA Institute Terms and Conditions

Figure 4 Maintenance 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 activation of (A) caspase-7 and (B) caspase-3. Active caspase-7 and caspase-3 (p20 and p17) were detected within 60 minutes after detachment of S-CECs. In P-CECs, activation of effector caspase-7 and caspase-3 was inhibited (data representative of 4 experiments). From 30 minutes on, P-CEC caspase-3–like activity was significantly lower than in S-CECs (C). n = 7; *P < .05, **P < .001. Gastroenterology 2007 132, 587-600DOI: (10.1053/j.gastro.2006.11.017) Copyright © 2007 AGA Institute Terms and Conditions

Figure 5 Inhibition of DNA fragmentation and chromatin condensation by preservation of cell-cell contacts. (A) DNA was extracted immediately after CEC isolation and 2 and 4 hours after pelleting (P-CEC, lanes 3–4) or suspension (S-CEC, lanes 5–6). Samples were analyzed by agarose gel electrophoresis. Cells incubated in suspension displayed intense DNA fragmentation, whereas pelleted cells did not. Lane 1, molecular mass marker (m). (B) At the indicated time points, P-CECs and S-CECs were fixed and stained with propidium iodine for DNA content and analyzed by flow cytometry. P-CECs contained considerably less fragmentated DNA than S-CECs did. Representative plots of 1 of 5 independent experiments are shown. (C) Sub-G1 proportions of S-CECs and P-CECs were quantified (n = 5). Sub-G1 peaks were significantly reduced in P-CECs. *P < .0005. (D) Freshly isolated CECs as well as S-CECs and P-CECs after 2 hours of incubation were fixed and nuclei were stained with 4′,6-diamidino-2-phenylindole. Only S-CECs displayed significant levels of chromatin condensation (original magnification 630×). Gastroenterology 2007 132, 587-600DOI: (10.1053/j.gastro.2006.11.017) Copyright © 2007 AGA Institute Terms and Conditions

Figure 6 Induction of anoikis after pellet incubation. Cytosolic extracts from S-CECs and P-CECs were prepared at the indicated times and analyzed for caspase-3–like activity. Additionally, P-CECs after 2 hours of incubation were resuspended in culture medium and further incubated in suspension (grey bars) before caspase-3–like activity was determined. In these cells, anoikis was induced to a similar extent than in freshly suspended CEC. n = 6, *P < .05, **P < .001. Gastroenterology 2007 132, 587-600DOI: (10.1053/j.gastro.2006.11.017) Copyright © 2007 AGA Institute Terms and Conditions

Figure 7 Antiapoptotic signals in P-CECs are FAK independent. Cytosolic extracts from S-CECs and P-CECs were analyzed for (A) FAK abundance and (B) FAK phosphorylation by Western blot at the indicated times. Intact FAK underwent 2 sequential cleavages in S-CECs, yielding fragments of 94/92 kilodaltons and 84 kilodaltons. In P-CECs, this cleavage was inhibited to a large extent. Dephosphorylation of FAK Tyr397 proceeded very rapidly in P-CECs while remaining stable in S-CECs for 60 minutes (data representative for 3 experiments). Gastroenterology 2007 132, 587-600DOI: (10.1053/j.gastro.2006.11.017) Copyright © 2007 AGA Institute Terms and Conditions

Figure 8 Akt, Erk1/2, EGFR, and Src remain phosphorylated in P-CECs. (A) Cellular aggregation preserves Akt activation. Cytosol was extracted from S-CECs and P-CECs at the indicated times and analyzed for phosphorylation of Akt. Akt phosphorylation remained stable for 2 hours in P-CECs but decreased during incubation in S-CECs. (B–D) Cytosolic extracts from P-CECs were analyzed for total levels and phosphorylation status of (B) Erk1/2, (C) EGFR, and (D) Src. Cell aggregation caused a transient increase in phosphorylation of EGFR and Erk1/2. Phosphorylation of Src was slightly decreased at the later time points, but Src was not completely deactivated (data representative of 3 experiments). Gastroenterology 2007 132, 587-600DOI: (10.1053/j.gastro.2006.11.017) Copyright © 2007 AGA Institute Terms and Conditions

Figure 9 Inhibition of Src and PI3-K reverses the effect of maintaining cell-cell contacts. Freshly isolated CECs were treated with increasing concentrations of (A) the MEK inhibitor U0126, (B) the EGFR inhibitor tyrphostin AG1478, (C) the PI3-K inhibitor LY294002, and (D) the Src inhibitor PP1, followed by centrifugation to form pellets. Cell aggregates were incubated for 2 hours at 37°C. Cytosol was extracted and analyzed for caspase-3–like activity. (A) Presence of U0126 had no influence on rates of P-CEC apoptosis (n = 12). (B and C) Incubation with tyrphostin AG1478 (n = 12) and LY294002 (n = 14) caused a dose-dependent increase in caspase-3–like activity. (D) PP1 induced a strong, dose-dependent increase in P-CEC apoptosis (n = 10). 0h, freshly isolated CEC; positive control, CEC without aggregation; *P < .05 compared with untreated cells; #P < .05 compared with positive control. Gastroenterology 2007 132, 587-600DOI: (10.1053/j.gastro.2006.11.017) Copyright © 2007 AGA Institute Terms and Conditions

Figure 10 Involvement of adherens junction proteins in the modulation of CEC anoikis. (A) β-catenin remains activated in P-CECs. Cytosolic extracts from S-CECs and P-CECs were analyzed at the indicated times for β-catenin content and its activation status. Total β-catenin levels declined in S-CECs but did not decline in P-CECs. Active β-catenin was completely lost in S-CECs after 120 minutes, whereas signals from active β-catenin were constant in P-CECs for 120 minutes (data representative of 3 experiments). (B) Modulation of anoikis by an agonist E-cadherin-Fc chimera protein. Freshly isolated CECs were incubated in suspension for 1 hour with E-cadherin-Fc chimera protein together with anti-Fc immunoglobulin G to induce aggregation or with anti-Fc immunoglobulin G alone (control). In the presence of E-cadherin-Fc, caspase-3–like activity was reduced by 25% (n = 7). Gastroenterology 2007 132, 587-600DOI: (10.1053/j.gastro.2006.11.017) Copyright © 2007 AGA Institute Terms and Conditions

Figure 11 Adhesion-mediated survival and anoikis in CECs. Cell-cell contact-mediated survival signaling can compensate for the absence of cell-matrix contacts. Preservation of cell-cell contacts induces dephosphorylation of FAK and maintains the activation status of Src, PI3-K, Akt, EGFR, and β-catenin. Only the loss of both cell-matrix and cell-cell contacts leads to the rapid onset of anoikis. Gastroenterology 2007 132, 587-600DOI: (10.1053/j.gastro.2006.11.017) Copyright © 2007 AGA Institute Terms and Conditions