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First published online 11 April 2006
doi: 10.1242/jcs.02894

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Caveolin-1 controls cell proliferation and cell death by suppressing expression of the inhibitor of apoptosis protein survivin

¹ Laboratory of Cellular Communications, FONDAP Center for Molecular Studies of the Cell (CEMC), Facultad de Medicina, Universidad de Chile, Av. Independencia 1027, Santiago, Chile
² Center of Medical Research Bruno Günther Building, School of Medicine, Universidad de Valparaíso, Valparaíso, Chile

View larger version (32K): [in a new window]   Fig. 1. Expression of caveolin-1 in HEK293T cells reduced proliferation and altered cell-cycle distribution. HEK293T cells were transfected with the plasmids pEGFP-C1, pEGFP-caveolin-1, pLacIOP or pLacIOP-caveolin-1 and plated 24 hours prior to measurements. Transfection efficiency in these experiments was roughly 50%. (A) Cell proliferation was measured by the MTS® assay. Values shown indicate the percentage of residual activity with respect to non-transfected controls. Black bars show viability of mock controls (pEGFP-C1 or pLacIOP) and hatched bars the viability of caveolin-1-transfected cells (pEGFP-caveolin-1 or pLacIOP-caveolin-1). Values were averaged from three independent experiments (mean ± s.e.m.). *Comparison with pEGFP-C1 control (P<0.001); #comparison with pLacIOP control (P<0.05). (B) Post-transfection with pEGFP-caveolin-1, the cell-cycle distribution of GFP-positive and GFP-negative HEK293T cells was compared by flow cytometry. Data shown were averaged from three independent experiments (see Table 1).

View larger version (14K): [in a new window]   Fig. 2. Expression of caveolin-1 in HEK293T cells promoted cell death. HEK293T cells were transfected with pLacIOP or pLacIOP-caveolin-1 as indicated and analyzed after 24 hours. (A) Viability was determined by the Trypan Blue exclusion assay. (B) To determine apoptosis, cells were harvested, resuspended in PBS with 10 µg/ml propidium iodide (PI) and analyzed by flow cytometry. In A and B values were averaged from three independent experiments (mean ± s.e.m.). #comparison with pLacIOP control (P<0.05), comparison with pLacIOP control (P<0.1). (C) Results from a standard DNA laddering assay. From left to right, samples from non-transfected (lane 1), pLacIOP-transfected (lane 2) and pLacIOP-caveolin-1-transfected (lane 3) cells are shown. (D) Nuclear fragmentation analysis. Caveolin-1 was detected by immunofluorescence (green) in cells transfected with pLacIOP-caveolin-1 and the integrity of the nuclei was assessed by PI staining (red). Samples were analyzed by confocal microscopy (Bar, 10 µm). In C and D a representative result from two independent experiments is shown.

View larger version (28K): [in a new window]   Fig. 3. Caveolin-1 expression in HEK293T cells reduced survivin mRNA and protein content. HEK293T cells were transfected with the plasmids pEGFP-C1, pEGFP-caveolin-1, pLacIOP or pLacIOP-caveolin-1 24 hours prior to measurements. (A) Cell extracts were analyzed by western blotting with anti-survivin, anti-caveolin-1 and anti-actin antibodies. From left to right: non-transfected cells (lane 1) and cells transfected with pEGFP-C1 (lane 2), pEGFP-caveolin-1 (lane 3), pLacIOP (lane 4) or pLacIOP-caveolin-1 (lane 5). Survivin levels were quantified by scanning densitometric analysis of western blots and normalized to actin. Residual survivin protein levels for pEGFP-caveolin-1 (0.55±0.06) and pLacIOP-caveolin-1 (0.49±0.05) transfected cells were compared to their respective mock controls. Numerical data represent the means ± s.e.m. of results obtained in three independent experiments; *comparison with their respective mock controls, P<0.001. (B) Alternatively, survivin was detected by immunofluorescence followed by FACS analysis in both green (GFP-positive) and non-green (GFP-negative) HEK293T cells, after transfection with the pEGFP-caveolin-1 vector. Data are representative of two experiments. (C) RT-PCR analysis of survivin. Actin was used as internal control. From left to right, samples from cells transfected with pEGFP-C1 (lane 1), pEGFP-caveolin-1 (lane 2), pLacIOP (lane 3) or pLacIOP-caveolin-1 (lane 4). Survivin mRNA levels for pEGFP-caveolin-1 (0.59±0.07)- and pLacIOP-caveolin-1 (0.50±0.07)-transfected cells were compared to their respective mock controls. Numerical data represent the means ± s.e.m. of results obtained in three independent experiments; *comparison with their respective mock controls, P<0.05.

View larger version (24K): [in a new window]   Fig. 4. Caveolin-1 suppressed ß-catenin-Tcf/Lef-dependent transcription of survivin in HEK293T cells. HEK293T cells were transfected with the plasmids pLacIOP or pLacIOP-caveolin-1 and subjected to further analysis after 24 hours. (A) Cellular localization of caveolin-1 (upper panels) and ß-catenin (middle panels) was detected by confocal microscopy; merged images (bottom panels). Bar, 10 µm. Note that co-localization was predominantly detected at the plasma membrane cell-cell contact sites (white arrows). (B) Ectopically expressed caveolin-1 was immunoprecipitated from HEK293T cells and then caveolin-1 and ß-catenin were detected by western blotting. A result representative of two independent experiments is shown. (C) HEK293T cells were co-transfected with the plasmids pLacIOP (vector) or pLacIOP-caveolin-1 (cav-1) and pTOP-FLASH (black bars) or pFOP-FLASH (white bars) in the presence or absence of 20 mM LiCl. After 24 hours, cell extracts were prepared and utilized for Tcf/Lef reporter assays (bar graph) and western blot analysis. Bar graph: luciferase activity (normalized to ß-galactosidase) as a percentage of the values detected in vector-transfected cells (pLacIOP, defined as 100%). Data shown are mean ± s.e.m. from three independent experiments; *comparison with pLacIOP control, P<0.01. Western blot: determination of survivin and actin levels. From left to right: cells transfected with pLacIOP (vector) or pLacIOP-caveolin-1 (cav-1) in the absence (-) or presence (+) of LiCl (20 mM). (D) HEK293T cells were co-transfected with the plasmids pLacIOP (black bars) or pLacIOP-caveolin-1 (hatched bars) and the reporter constructs pLuc-1710, pLuc-420, pLuc-420-2M or pLuc-420-3M as indicated. After 24 hours, cell extracts were prepared and used to measure reporter activity. Data shown are mean ± s.e.m. from three independent experiments; #comparison with pLuc-1710 in the presence of pLacIOP, P<0.001; comparison with pLuc-420 in the presence of pLacIOP, P<0.01.

View larger version (35K): [in a new window]   Fig. 5. Ectopic expression of GFP-survivin in HEK293T cells reversed the reduction in cell proliferation and changes in the cell cycle caused by caveolin-1. (A) HEK293T cells were transfected with pLacIOP alone (3 µg) or co-transfected with the vectors pLacIOP-caveolin-1 (3 µg) and increasing amounts of the vector pEGFP-survivin (up to 2 µg). Controls included transfections with pEGFP-C1 (2 µg) or pEGFP-survivin (2 µg). Cell proliferation was measured by the MTS® assay. Values detected following transfection are shown as a percentage of the MTS activity measured in non-transfected controls (100%). (B) HEK293T cells were either transfected with pEGFP-caveolin-1 alone (5 µg) or co-transfected with the vectors pEGFP-caveolin-1 (5 µg) and pEGFP-survivin (5 µg) and cell cycle was analyzed by flow cytometry 48 hours post-transfection by gating on GFP-negative (lane 1) and GFP-positive (lanes 2 and 3) subpopulations. GFP-caveolin-1 and GFP-survivin expression are shown in a representative western blot. A result representative of two independent experiments is shown. (C) HEK293T cells were transfected with the vector pLacIOP (3 µg) alone or co-transfected with the vectors pLacIOP-caveolin-1 (3 µg) and increasing amounts of pEGFP-survivin (up to 2 µg) and cultured post-transfection in the presence of either 250 nM doxorrubicin, 25 µM etoposide or 250 nM taxol. Cell proliferation was evaluated by the MTS® assay. Values shown are equivalent to the percentage of residual activity as compared with non-transfected controls (100%). In A and C the data shown are mean ± s.e.m. from three independent experiments; *comparison with pLacIOP control, P<0.01; #comparison with pLacIOP-caveolin-1, P<0.01.

View larger version (14K): [in a new window]   Fig. 6. Ectopic expression of caveolin-1 in ZR75 human breast cancer cells downregulated survivin and decreased cell proliferation. ZR75 cells were stably transfected with the plasmids pLacIOP (-) or pLacIOP-caveolin-1 (+). (A) Survivin and caveolin-1 expression was assessed by western blotting. A representative result is shown. Residual survivin protein levels were 0.53±0.09 as determined in three independent experiments (*P<0.05). (B) Survivin mRNA content was analyzed by RT-PCR. Residual survivin mRNA levels were 0.50±0.06 as averaged from three independent experiments (*P<0.05). (C) Cell proliferation was measured by the MTS® assay in the presence and absence of caveolin-1. (D) Apoptosis was determined as indicated above by PI staining followed by flow cytometry. Numerical results are mean ± s.e.m. from three independent experiments; # and comparisons with the mock control pLacIOP, #P<0.01, P<0.05.

View larger version (35K): [in a new window]   Fig. 7. Downregulation of caveolin-1 by si-RNA in NIH3T3 cells increased survivin expression. (A) NIH3T3 cells were transfected with caveolin-1-specific siRNA or control siRNA, as described. Expression of caveolin-1, survivin, actin and caveolin-2 was assessed by western blotting. A result representative of three independent experiments is shown. (B) Caveolin-1 and survivin levels were quantified by scanning densitometry. The ratios for caveolin-1 to actin (black bar), caveolin-2 to actin (white bar) and survivin to actin are shown as a percentage of the values obtained in control experiments. # and comparisons with their respective controls, *P<0.01, #P<0.05). (C) Cell proliferation was measured by the MTS® assay. (D) Tcf/Lef reporter assay. NIH3T3 cells were transfected with caveolin-1-specific siRNA or the control siRNA and grown for 24 hours. Then, cells were additionally transfected with the plasmid pTOP-FLASH. After another 24 hours, luciferase activity was determined and normalized to ß-galactosidase activity, as described above. All values are mean ± s.e.m. from either three (B) or two (C,D) independent experiments.

View larger version (28K): [in a new window]   Fig. 8. Caveolin-1 downregulated survivin expression in cells with enhanced or constitutive activity via the Wnt pathway. (A) HEK293T cells were transfected with either pLacIOP (-) or pLacIOP-caveolin-1 (+) and then cultured either in normal medium (control) or conditioned medium containing Wnt3a for 24 hours (see Materials and Methods for details). Survivin, caveolin-1 and actin protein levels were assessed by western blotting. (B) Clones of DLD1 colon adenocarcinoma cells stably transfected with either pLacIOP (M1) or pLacIOP-caveolin-1 (C4) were described previously (Bender et al., 2000). Cell extracts were prepared post-induction with 1 mM IPTG for 24 hours. Survivin, caveolin-1 and actin expression were assessed by western blotting. Residual survivin level was 0.39±0.10 (mean ± s.e.m.; *P<0.05), as determined in three independent experiments. (C) Survivin mRNA content in DLD1 clones was analyzed by RT-PCR. Residual survivin mRNA level was 0.49±0.18 (mean ± s.e.m.; #P<0.05), as determined in three independent experiments.

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