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First published online 15 March 2005
doi: 10.1242/jcs.02278

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Caveolin-1 mediates the expression and localization of cathepsin B, pro-urokinase plasminogen activator and their cell-surface receptors in human colorectal carcinoma cells

¹ Department of Pharmacology, Wayne State University, School of Medicine, Detroit, MI 48201, USA
² Barbara Ann Karmanos Cancer Institute, Wayne State University, School of Medicine, Detroit, MI 48201, USA

View larger version (43K): [in a new window]   Fig. 1. Establishment of a stable antisense caveolin-1 HCT 116 human colorectal-carcinoma cell line. HCT 116 cells were transfected with either empty vector (control) or vector containing caveolin-1-encoding cDNA in the antisense orientation (AS-cav-1) and stable clones were selected in the presence of 0.5 mg ml–1 puromycin for 15 days. (A) Parental, control and AS-cav-1 HCT 116 cells were solubilized in lysis buffer containing 1% Triton X-100, 60 mM octylglucoside. 20 µg protein were analysed by SDS-PAGE and immunoblotted with antibodies to human caveolin-1 and human ß-actin in order to verify equal loading. Immunoblots are shown in triplicate and are representative of at least three experiments. (B) 1 µl total RNA isolated from control and AS-cav-1 HCT 116 cells was subjected to reverse transcription followed by PCR using primer sequences to caveolin-1 and ß2-microglobulin (as a control). PCR products were resolved on a 1.2% agarose gel stained with ethidium bromide. (C) Equal amounts of protein for HCT 116, control and AS-cav-1 cells were subjected to subcellular fractionation on a sucrose gradient after homogenization in sodium carbonate buffer, pH 11.0. Fractions were collected from the top of the gradient and equal volume aliquots of fractions 2-11 were analysed by SDS-PAGE and immunoblotted with anti-human caveolin-1 antibodies. Immunoblots are representative of at least three experiments. (D) Staining was performed on control and AS-cav-1 HCT 116 cells grown on glass coverslips. Cells were incubated in the presence of saponin with primary antibodies to rabbit anti-human caveolin-1 IgG and then incubated with Texas-Red-conjugated affinity-purified donkey anti-rabbit IgG plus normal donkey serum. The cells were observed with a Zeiss LSM 310 microscope in confocal mode at a magnification of 630 x under oil immersion. Staining for caveolin-1 is shown in red. Bar, 10 µm.

View larger version (36K): [in a new window]   Fig. 2. Differences in the expression, localization and activity of cathepsin B in AS-cav-1 HCT 116 cells. (A) 1 µl total RNA isolated from control and AS-cav-1 HCT 116 cells was subjected to reverse transcription followed by PCR using primer sequences to cathepsin B and ß2-microglobulin (as a control). PCR products were resolved on a 1.2% agarose gel stained with ethidium bromide. (B) Conditioned media from control and AS-cav-1 HCT 116 cells were collected and the cells were solubilized in lysis buffer containing 0.1% Triton X-100. Media and cell lysates (40 µg protein) were analysed by SDS-PAGE and immunoblotted with anti-human-cathepsin-B antibodies. Immunoblots are representative of at least three experiments. (C) Conditioned media and cell lysates from control and AS-cav-1 HCT 116 cells were assayed for cathepsin-B activity. Intracellular (cell lysates) and extracellular (media) cathepsin-B activities were measured against Z-Arg-Arg-NHMec substrate and activity was expressed as pmoles min–1 (µg DNA)–1. The graphs are representative of at least three experiments and presented as means ± standard deviation (s.d.). **P<0.01. (D) Equal amounts of protein for control and AS-cav-1 HCT 116 cells were subjected to subcellular fractionation on a sucrose gradient after homogenization in sodium-carbonate buffer, pH 11.0. Fractions were collected from the top of the gradient and equal-volume aliquots of fractions 2-11 were analysed by SDS-PAGE and immunoblotted with anti-human-cathepsin-B antibody. Immunoblots are representative of at least three experiments.

View larger version (43K): [in a new window]   Fig. 3. Expression and localization of p11 to caveolae are reduced in AS-cav-1 HCT 116 cells. (A) 1 µl total RNA isolated from control and AS-cav-1 HCT 116 cells was subjected to reverse transcription followed by PCR using primer sequences to p11 and ß2-microglobulin (as a control). PCR products were resolved on a 1.2% agarose gel stained with ethidium bromide. (B) Control and AS-cav-1 HCT 116 cells were solubilized in lysis buffer containing 1% Triton X-100 and 60 mM octylglucoside. 25 µg protein were analysed by SDS-PAGE and immunoblotted with anti-human-p11 antibody. Immunoblots are shown in triplicate and are representative of at least three experiments. (C) Equal amounts of protein for control and AS-cav-1 HCT 116 cells were subjected to subcellular fractionation on a sucrose gradient after homogenization in sodium-carbonate buffer, pH 11.0. Fractions were collected from the top of the gradient and equal-volume aliquots of fractions 2-11 were analysed by SDS-PAGE and immunoblotted with anti-human-p11 antibodies. Immunoblots are representative of at least three experiments.

View larger version (47K): [in a new window]   Fig. 4. Expression, secretion and localization of pro-uPA to caveolae are downregulated in AS-cav-1 HCT 116 cells. (A) Conditioned media from control and AS-cav-1 HCT 116 cells were collected and the cells were solubilized in lysis buffer containing 1% Triton X-100, 60 mM octylglucoside. Media and cell lysates (25 µg protein) were analysed by SDS-PAGE and immunoblotted with anti-human-uPA antibodies. Immunoblots are shown in triplicate and are representative of at least three experiments. (B) Equal amounts of protein for control and AS-cav-1 HCT 116 cells were subjected to subcellular fractionation on a sucrose gradient after homogenization in sodium-carbonate buffer, pH 11.0. Fractions were collected from the top of the gradient and equal-volume aliquots of fractions 2-11 were analysed by SDS-PAGE and immunoblotted with anti-human-uPA antibodies. Immunoblots are representative of at least three experiments.

View larger version (38K): [in a new window]   Fig. 5. Expression and localization of uPAR and ß1-integrin to caveolae are downregulated in AS-cav-1 HCT 116 cells. (A) Control and AS-cav-1 HCT 116 cells were solubilized in lysis buffer containing 1% Triton X-100, 60 mM octylglucoside. 30 µg protein were analysed by SDS-PAGE and immunoblotted with anti-human-uPAR antibodies. Immunoblots are shown in triplicate and are representative of at least three experiments. (B) Equal amounts of protein for control and AS-cav-1 HCT 116 cells were subjected to subcellular fractionation on a sucrose gradient after homogenization in sodium-carbonate buffer, pH 11.0. Fractions were collected from the top of the gradient and equal-volume aliquots of fractions 2-11 were analysed by SDS-PAGE and immunoblotted with anti-human-uPAR antibody. (C) Control and AS-cav-1 HCT 116 cells were solubilized in lysis buffer containing 1% Triton X-100, 60 mM octylglucoside. 20 µg protein were analysed by SDS-PAGE and immunoblotted with anti-human-ß1-integrin antibody. (D) Equal amounts of protein for control and AS-cav-1 HCT 116 cells were subjected to subcellular fractionation on a sucrose gradient after homogenization in sodium-carbonate buffer, pH 11.0. Fractions were collected from the top of the gradient and equal-volume aliquots of fractions 2-11 were analysed by SDS-PAGE and immunoblotted with anti-human-ß1-integrin antibodies. Immunoblots are representative of at least three experiments.

View larger version (62K): [in a new window]   Fig. 6. Spheroids of live AS-cav-1 HCT 116 cells degrade less DQ-collagen IV both intracellularly (long arrow) and pericellularly (arrowhead) than do spheroids of live control HCT 116 cells. (A) Cells were plated on DQ-collagen-IV/Matrigel-coated coverslips and imaged at 48 hours. Fluorescent images of DQ-collagen-IV degradation products (green), taken using an extended depth of focus, are superimposed on phase images of live spheroids of control and AS-cav-1 HCT 116 cells. Bar, 10 µm. Images are representative of at least three experiments. (B) Fluorescent intensity (intracellular plus pericellular) was measured using Metamorph 6.0 software and is expressed as average intensity. The graph is representative of at least three experiments and is presented as mean ± s.d. **P<0.01.

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