Ectopic caveolin-1 expression reduced survivin mRNA and protein levels in HT29(ATCC) but not HT29(US) human colon cancer cells. Results obtained with HT29(ATCC) cells (A, B, and C) and HT29(US) cells (D, E, and F) stably transfected with the plasmids pLacIOP and pLacIOP-caveolin-1 are shown. Cells were grown for 24 h in the absence (−) or presence (+) of 1 mM IPTG. (A and D) Total protein extracts from HT29(ATCC) or HT29(US) cells were separated by SDS-PAGE (50 μg total protein per lane) and analyzed by Western blotting with anti-caveolin-1, antisurvivin, and antiactin antibodies. Survivin levels were quantified by scanning densitometric analysis of Western blots and normalized to actin. Survivin levels in cells with IPTG, pLacIOP-caveolin-1, or both were compared to those in the pLacIOP no-IPTG control. Data are means ± SEM for three independent experiments. (B and E) Survivin mRNA levels were assessed by semiquantitative RT-PCR analysis in cells grown in the absence (−) or presence (+) of 1 mM IPTG. Actin was used as an internal control. Data are means ± SEM for three independent experiments. Survivin mRNA content in cells with IPTG, pLacIOP-caveolin-1, or both were compared to the pLacIOP no-IPTG control. (C and F) Survivin mRNA levels were determined by QPCR analysis in cells grown as indicated for panels B and E. 18S rRNA was used as an internal control. Data are means ± SEM for three independent experiments. Statistically significant differences compared with the pLacIOP no-IPTG control are indicated (*, P < 0.01; #, P < 0.05).

Localization of β-catenin and caveolin-1 in HT29(ATCC) and HT29(US) cells expressing caveolin-1. (A) HT29(ATCC), HT29(US), and MDCK cells were grown on glass coverslips and incubated for 24 h in the presence of 1 mM IPTG. Samples were analyzed by indirect immunofluorescence and confocal microscopy. Cellular localization of caveolin-1 (cav-1, green) and β-catenin (β-cat, red) is shown. White arrows indicate areas corresponding to insets (bottom row), shown at a higher magnification. Merged images were generated with Imaris software, whereby a third pseudocolor channel (white) was used to show colocalization of red and green pixels. The software provides an automated selection of colocalization based on previously described algorithms (8). Bar, 10 μm. (B and C) Proteins (50 μg per lane) from HT29(ATCC) cells (B) and HT29(US) cells (C) transfected with pLacIOP or pLacIOP-caveolin-1 were separated by SDS-PAGE, and the presence of E-cadherin, β-catenin, caveolin-1, and actin was assessed by Western blotting.

Characterization of clonal HT29(ATCC) cell populations expressing caveolin-1. (A) Clonal HT29(ATCC) cell populations stably transfected with pLacIOP-caveolin-1 were isolated as described in Materials and Methods from the parental HT29(ATCC) pLacIOP-caveolin-1-transfected population (batch). The pLacIOP- and pLacIOP-caveolin-1 batch-transfected cells, as well as clonal cell populations (clones 1 and 2), were cultured for 24 h in the presence of 1 mM IPTG. Total protein extracts (50 μg per lane) were separated by SDS-PAGE and analyzed by Western blotting for the presence of E-cadherin (E-cad), β-catenin (β-cat), caveolin-1 (cav-1), survivin, and actin using specific antibodies. (B) Survivin mRNA levels were determined by QPCR in the stably transfected HT29(ATCC) cell populations used for panel A and expressed as a percentage of the value for nontransfected cells. 18S rRNA was used as an internal control. Statistically significant differences compared with the pLacIOP vector are indicated (#, P < 0.05). (C) HT29(ATCC) cell populations analyzed for panel A were also transfected with pTOP-FLASH and pFOP-FLASH (2 μg each). After 24 h, cell extracts were obtained and luciferase activity was determined. Values for luciferase activity were standardized by calculating the TOP/FOP activity ratio for each condition. The data are means ± SEM from two independent experiments in triplicate. Statistically significant differences compared with pLacIOP vector alone are indicated (*, P < 0.01).

Caveolin-1 and E-cadherin cooperated to inhibit β-catenin-Tcf/Lef-dependent transcription. (A) HEK293T cells were either transfected with pLacIOP-caveolin-1 alone (2 μg) or cotransfected with pLacIOP-caveolin-1 (2 μg) and pBATEM2 (4 μg). After 24 h in the presence of 1 mM IPTG, caveolin-1 (cav-1), β-catenin (β-cat), and E-cadherin (E-cad) protein levels were assessed in caveolin-1 immunoprecipitates by Western blotting. (B) HEK293T cells were cotransfected with pTOP-FLASH and pFOP-FLASH and with pLacIOP or pLacIOP-caveolin-1 and in the absence or presence of pBATEM2, coding for E-cadherin. For transfection experiments, 2 μg of each plasmid was employed. After 24 h in the presence of 1 mM IPTG, cell extracts were obtained and luciferase activity was determined. Values for luciferase activity were standardized by calculating TOP/FOP activity ratios for each condition. The data are means ± SEM from two independent experiments in triplicate. Statistically significant differences are indicated (#, P < 0.05).

E-cadherin promoted the association of caveolin-1 with β-catenin in HT29(US) cells. HT29(US) cells transfected with pLacIOP-caveolin-1 (cav-1) or HT29(US) cells cotransfected with pLacIOP-caveolin-1 and pBATEM2 (cav-1 + E-cad) were cultured for 24 h in the presence of 1 mM IPTG. (A) Cell extracts were obtained and caveolin-1 was immunoprecipitated. Thereafter, E-cadherin (E-cad) and β-catenin (β-cat) levels were assessed in caveolin-1 (cav-1) immunoprecipitates by Western blotting. (B) Caveolin-1, β-catenin, and E-cadherin localization was assessed by indirect immunofluorescence and confocal microscopy. White arrows indicate areas shown in insets at higher magnifications. Merged images were generated with Imaris software as described for Fig. 3. Bar, 10 μm.

Expression of E-cadherin in the metastatic melanoma B16-F10 cell line facilitated caveolin-1-mediated down-regulation of survivin and cell cycle control. B16-F10 murine melanoma cells stably transfected with either pLacIOP or pLacIOP-caveolin-1 were grown for 24 h in complete medium and then transfected with the plasmid pBATEM2 by electroporation, as described in Materials and Methods. Thereafter, cells were grown in the presence of 1 mM IPTG for 48 h and then subjected to analysis. (A) Cell extracts were obtained and analyzed by Western blotting. A result representative of three independent experiments is shown. Residual survivin levels in cells transfected with pLacIOP-caveolin-1, pLacIOP/pBATEM2, or pLacIOP-caveolin-1/pBATEM2 were compared to levels in cells transfected with pLacIOP alone (control). Data are means ± SEM for three independent experiments. Statistically significant differences compared with the pLacIOP control are indicated (*, P < 0.01). (B) Cell proliferation was measured by the MTS assay. Data were averaged from three independent experiments (means ± SEM; #, P < 0.05). (C) Cell cycle distribution was assessed by flow cytometry. Data are representative of three independent experiments. (D) To determine apoptosis, cells were harvested, resuspended in PBS with 10 μg/ml PI, and analyzed by flow cytometry. Data are means ± SEM from three independent experiments. Statistically significant differences detected with an unpaired (#, P < 0.05) or paired (&, P < 0.01) t test are indicated.

Expression and localization of β-catenin and E-cadherin in HT29(ATCC) and HT29(US) colon cancer cells. (A) HT29(ATCC), HT29(US), and MDCK cells were cultured for 48 h and then harvested. Protein extracts were prepared, separated by SDS-PAGE (50 μg total protein per lane), and analyzed by Western blotting with anti-E-cadherin, anti-caveolin-1, and antiactin antibodies. Bands corresponding to caveolin-1α and -1β are indicated. (B) HT29(ATCC) and HT29(US) cells were grown for 48 h on glass coverslips, and samples were analyzed by indirect immunofluorescence and confocal microscopy. Cellular localization of β-catenin (β-cat) and E-cadherin (E-cad) are shown in green, while the nucleus was stained with PI. Results are representative of two independent experiments. White arrows indicate areas corresponding to insets showing merged images at a higher magnification. Bar, 10 μm.

Ectopic caveolin-1 expression reduced the nuclear presence of β-catenin in HT29(ATCC) cells. HT29(ATCC) cells transfected with pLacIOP (A and C) or pLacIOP-caveolin-1 (B and D) were stained with anti-β-catenin antibodies (green) and PI (red). A confocal optical section through the center of the cell is shown for cells lacking (A) or expressing (B) caveolin-1. Bar, 7 μm. Images in panels C and D correspond to those in A and B, respectively, after processing with Imaris software. The laser confocal image z stack was visualized three-dimensionally using the IsoSurface processing mode, which creates an artificial real-volume object from available voxels. The z axis of the group of cells was rotated to the position indicated (yellow line), and a virtual section through the images was generated at the center of the cells so that the green label inside the nucleus could be clearly identified. (E) HT29(ATCC) and HT29(US) cells transfected with pLacIOP or pLacIOP-caveolin-1 were processed with a proteomic extraction system kit following the manufacturer's instructions to yield subcellular fractions corresponding to cytosol (1), plasma membrane/organelles (2), nucleus (3), and cytoskeleton (4). Proteins from equal volumes of each fraction were concentrated, separated by SDS-PAGE, and analyzed by Western blotting with anti-β-catenin and anti-cyclin D1 antibodies. Cyclin D1 was utilized here as an internal control for the nuclear fraction. (F) HT29(ATCC) cells were cotransfected with 2 μg of pTOP-FLASH, pFOP-FLASH, pLacIOP, or pLacIOP-caveolin-1 and cultured for 24 h in the absence (−) or presence (+) of 1 mM IPTG. Then, cell extracts were prepared and luciferase activity was determined as described in Materials and Methods. Values for luciferase activity were standardized by calculating the TOP/FOP activity ratio for each condition. Data are means ± SEM for two independent experiments in triplicate. Statistically significant differences compared with pLacIOP vector alone are indicated (*, P < 0.01).

Caveolin-1 associated with β-catenin and E-cadherin in several cell lines. (A) HT29(US), DLD1, and HT29(ATCC) cells stably transfected with pLacIOP-caveolin-1 were cultured in presence of 1 mM IPTG for 24 h. MDCK cells at confluence after 48 h in culture were included as a control for endogenous caveolin-1. HEK293T cells were transiently transfected with pLacIOP-caveolin-1 and analyzed 24 h posttransfection in the presence of 1 mM IPTG. Endogenous or ectopically expressed caveolin-1 was immunoprecipitated from cell extracts (500 μg total proteins). Results obtained with an unrelated antibody (IgG) were included as a negative control. Then caveolin-1 (cav-1), β-catenin (β-cat), and E-cadherin (E-cad) presence was detected by Western blotting. Numerical values shown below individual blots indicate the percentage of total β-catenin averaged from two independent experiments in the respective cell extracts that coimmunoprecipitated with caveolin-1 corrected in each case for efficiency of caveolin-1 immunoprecipitation (percent recovery). (B) HT29(ATCC), HT29(US), and DLD1 cells transfected with pLacIOP vector were cultured in the presence of 1 mM IPTG, and protein extracts were subsequently immunoprecipitated using the conditions described above. Immunoprecipitation of an extract of DLD1 cells stably transfected with pLacIOP-caveolin-1 and cultured in the presence of IPTG was utilized as a positive control.

Coexpression of E-cadherin and caveolin-1 in HT29(US) cells. HT29(US) cells were cotransfected with pBATEM2 and either pLacIOP or pLacIOP-caveolin-1. Stably transfected cells were selected in the presence of hygromycin as described in the text. These cells were compared to HT29(US) cells transfected with pLacIOP or pLacIOP-caveolin-1. (A) Cell extracts were obtained from each cell line 48 h after plating. Proteins were separated by SDS-PAGE (50 μg total protein per lane) and analyzed by Western blotting using antibodies specific for E-cadherin (E-cad), caveolin-1 (cav-1), and actin. (B) Cells were grown for 48 h in the presence of 1 mM IPTG, harvested, fixed, and prepared for detection of E-cadherin using a monoclonal antibody and a FITC-coupled second antibody. E-cadherin-specific cell surface staining was visualized by flow cytometry for cells expressing caveolin-1 alone (not shown), E-cadherin (gray line), or caveolin-1 and E-cadherin (black area). Results for a negative control (cells without first antibody) are also shown; the fluorescence was similar to that for caveolin-1 expressed alone (not shown). A representative result from two independent experiments is shown in each panel.

Re-expression of E-cadherin in HT29(US) cells restored caveolin-1-mediated inhibition of survivin expression and cell proliferation. (A) HT29(US) cells stably cotransfected with pBATEM2 and pLacIOP or pLacIOP-caveolin-1 were grown for 24 h in the absence (−) or presence (+) of 1 mM IPTG. Then, cell extracts were obtained for analysis. Following protein separation by SDS-PAGE (50 μg total protein per lane), E-cadherin (E-cad), caveolin-1 (cav-1), survivin, and actin levels were assessed by Western blotting. Survivin levels were quantified by scanning densitometric analysis of Western blots and normalized to actin. Survivin levels in cells with IPTG, pLacIOP-caveolin-1, or both were compared to those in the pLacIOP no-IPTG control. Data are means ± SEM for three independent experiments. Statistically significant differences compared with the control are indicated (*, P < 0.01). (B) Survivin mRNA levels were assessed by QPCR analysis in HT29(US) cells stably cotransfected as indicated above. 18S rRNA was used as an internal control. Statistically significant differences compared with the control are indicated (#, P < 0.05). (C) Cell proliferation was measured by the MTS assay in HT29(US) cells stably transfected with pBATEM2 (E-cad) and either pLacIOP (−cav1) or pLacIOP-caveolin-1 (+cav1). Here, IPTG was present at 1 mM in all four conditions evaluated. Data are means ± SEM from two independent experiments in triplicate. Statistically significant differences are indicated (#, P < 0.05). (D) HT29(US) cells were transfected with the plasmids used for panel C together with pTOP-FLASH and pFOP-FLASH (2 μg each). After 24 h, cell extracts were obtained and luciferase activity was determined. Values for luciferase activity were standardized by calculating the TOP/FOP activity ratio for each condition. Data are means ± SEM from two independent experiments in triplicate. Statistically significant differences are indicated (*, P < 0.01).