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First published online 30 July 2003
doi: 10.1242/jcs.00644

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Bcl-2 expression decreases cadherin-mediated cell-cell adhesion

Department of Cell Biology, University of Alberta, Edmonton, Alberta T6G2H7, Canada

View larger version (162K): [in a new window]   Fig. 1. Phase contrast image of confluent cultures (A,B) and electron micrograph images showing cell structure (C,D) of MCF-7 (A,C) and MCF-7B (B,D) cells. TJ, tight junction; AJ, adherens junction; Des, desmosome. (A,B) Magnification, 250x. Scale bars: (C) 0.42 µm; (D), 0.21 µm

View larger version (122K): [in a new window]   Fig. 2. Subcellular localization of Bcl-2 and components of the adherens junction, tight junction and desmosomes in MCF-7 and MCF-7B cells. Cultures were grown on glass coverslips to confluence, formaldehyde fixed, CSK permeabilized and stained with antibodies to Bcl-2, and the indicated junctional proteins at concentrations listed in Table 1. Primary antibodies were detected by FITC- or rhodamine-conjugated species-specific secondary antibodies and viewed with a 100x objective using a BX50 Olympus microscope. Images were captured using Advance Spot software. Scale bar: 50 µm.

View larger version (111K): [in a new window]   Fig. 3. Reorganization of the junctional proteins in MCF-7 and MCF-7B cells grown in the absence of estrogen. Cells were grown to confluence on glass coverslips, transferred to estrogen-depleted medium and maintained for another 8 days. Cells were fixed and processed for indirect IF as outlined in the legend of Fig. 2. Scale bar: 50 µm.

View larger version (203K): [in a new window]   Fig. 4 (A,B) Membrane redistribution of junctional proteins in MCF-7B cells upon long-term absence of estrogen. Coverslip-grown confluent cultures were transferred into estrogen-deprived medium for 16 days. IF localization of Bcl-2 and various junctional proteins was carried out as outlined in the legend of Fig. 2. Double staining was performed by detecting two different primary antibodies with FITC- or rhodamine-conjugated species-specific secondary antibodies. Merged images (right column, A: bottom row, B) were constructed using the Adobe Photoshop software. CK, cytokeratins; Pg, plakoglobin. Arrows in A indicate the absence of Bcl-2 expression or membrane localization of junctional proteins. Scale bars: (A) 150 µm; (B) 75 µm

View larger version (67K): [in a new window]   Fig. 5. Bcl-2 over expression modifies the level of junctional proteins and decreases the surface E-cad. (A) MCF-7 and MCF-7B cells were grown to confluence in normal medium (day 0). Replicate cultures were transferred into estrogen-free medium for 2 to 16 days. Equal amounts of TCEs from control and estrogen-depleted cultures was resolved on 10% (Bcl-2 and tubulin) or 6% (E-cad, -cat, ß-cat, Pg, p120 and ZO-1) gels and processed for IB with various antibodies at concentrations listed in Table 1. (B) MCF-7 and MCF-7B were grown in normal medium and duplicate cultures were transferred into estrogen-deprived media for 12 days. Cultures were biotinylated as described in Materials and Methods and TCEs prepared. From each line, 250 µg of TCE was precipitated with streptavidin-agarose beads. Biotinlylated proteins were eluted from the beads and, together with 50 µg of TCEs, processed for IB with E-cad antibodies. To verify comparable loading the TCE blot was reprobed for tubulin. (C) Confluent MCF-7 and MCF-7B cells were either fixed and then permeabilized (Total) or CSK-extracted and then fixed (Surface) and processed for staining with an E-cad antibody (Transduction Laboratories). Scale bar: 50 µm.

View larger version (54K): [in a new window]   Fig. 6. Disruption and intracellular redistribution of ZO-1 coincide with increased levels of ErbB2 in MCF-7B cells. (A) Cultures were established on glass coverslips, formaldehyde-fixed, CSK permeabilized and processed for IF staining with ErbB2 and ZO-1 antibodies. Scale bar: 50 µm. (B) TCEs were prepared for confluent MCF-7, MCF-7B and SKBR-3 cultures and 50 µg from each line processed for IB with ErbB2 antibodies.

View larger version (79K): [in a new window]   Fig. 7. Bcl-2 expressed in MDCK cells inhibits junction formation and co-distributes with the soluble ZO-1. (A) Ultrastructural organization of uninduced and Zn2+-induced Bcl-2-expressing MDCK cells. MDCK-Bcl-2 transfectants were grown on collagen- and carbon-coated glass coverslips without (left) or with (right) Zn2+ and processed for electron microscopy. (B) Confluent Zn2+-induced, MDCK-Bcl-2 transfectants grown on coverslips were processed for double IF with a mixture of Bcl-2 and ZO-1 antibodies. Primary antibodies were detected with species-specific secondary antibodies and viewed with a 63x objective. (C) Duplicate cultures from B were either formaldehyde fixed, CSK-permeabilized (Fixed) or CSK extracted first and then fixed (Extracted) and processed for Bcl-2 and ZO-1 staining. (D) Duplicate cultures from B were formaldehyde fixed, CSK-permeabilized and processed for double IF staining with Bcl-2 and p120 antibodies. Scale bars: 1.25 µm (A); 85 µm (B); 40 µm (C); 50 µm (D).

View larger version (68K): [in a new window]   Fig. 8. Bcl-2 expression in MDCK cells decreases the cytoskeleton-associated pool of the junctional proteins and the surface E-cad. (A) Confluent, vector transfected (Neo) and two Zn2+-induced independent MDCK-Bcl-2 transfectants (B1, B2) were extracted with CSK buffer and the soluble (S) and insoluble (P) fractions separated. Fractions were resolved on 10% (Bcl-2 and tubulin) or 6% (E-cad, -cat, ß-cat, Pg, and ZO-1) gels and processed for IB with various antibodies. This experiment was repeated 5 times producing similar results with very little variability among the experiments. (B) The protein bands in A were scanned and quantitated using the NIH imager software. The value obtained for each protein/cell line was normalized to the value obtained for tubulin in the same lysate/cell line and the ratio of the soluble to insoluble calculated. (C) Confluent MDCK-Neo and Zn2+-induced MDCK-B1 and MDCK-B2 cultures were biotinylated and equivalent amounts of total proteins precipitated by streptavidinagarose beads. Biotinylated complexes were eluted, separated on SDS/6% gels and together with 50 µg of the TCE from each line were processed for IB with E-cad antibodies. To confirm equal loading, the TCE blots were reprobed with tubulin antibodies. (D) Confluent MDCK-Neo and Zn2+-induced MDCK-Bcl-2 cells were either fixed and then permeabilized (Total) or CSK-extracted and then fixed (Surface) and processed for staining with an E-cad antibody (3G8). Scale bar: 50 µm.

View larger version (13K): [in a new window]   Fig. 9. Decreased transepithelial resistance (TER) in Bcl-2 expressing MDCK and MCF-7B cultures. Confluent MDCK Neo, Zn2+-induced MDCK-B1 and MDCK-B2, MCF-7 and MCF-7B cells were grown on filters in normal medium (1.8 mM calcium, HCM) as described in Materials and Methods. Duplicate MDCK Neo cultures were transferred into LCM for 3 hours and TER measurements were performed on all filters. Histograms represent the mean±s.e.m. of three independent measurements for each cell line. The absence of error bars indicates the small differences in the three measurements.