Article Figures & Data
Figures
- FIGURE 1.
Density dependence of scp-2 cells expressing activated M-Ras plated in serum-free medium. GFP scp-2 or G22V M-Ras scp-2 cells were plated at 2.5 × 104 or 5 × 104 cells/ml in DMEM with no FBS or exogenous growth factors and cultured for 4 days. A. Photomicrographs of cultures at day 4. Bar, 40 μm. B. Counts of viable cells. Columns, mean; bars, SD. Results are representative of four experiments.
- FIGURE 2.
Medium conditioned by scp-2 cells expressing activated G22V M-Ras maintained numbers of viable G22V M-Ras scp-2 cells. GFP scp-2 cells (open bars) or G22V M-Ras scp-2 cells (solid bars) were plated at the low cell density of 1.25 × 104 cells/ml in 5 ml DMEM in the absence of FBS and exogenous growth factors, with or without the addition to the final percentages shown, of medium conditioned by G22V M-Ras scp-2 cells. At day 4, cells were harvested and viable cells were counted. Results are representative of two experiments.
- FIGURE 3.
Density dependence of anchorage-independent growth of scp-2 cells expressing activated G22V M-Ras. G22V M-Ras scp-2 cells were cultured at either 103 or 104 cells/ml as indicated in 1 ml DMEM supplemented with 0.3% agar and 5% FBS with or without the addition of CM from G22V M-Ras scp-2 cells adjusted to a final concentration of 50%. At day 14, the colonies were photographed (A) or counted (B). A. Photomicrographs of colonies grown at the indicated cell densities with or without CM from G22V M-Ras scp-2 cells. B. Cloning efficiency is defined as the number of colonies formed as a percentage of the total number of cells plated. Columns, mean; bars, SD. Results are representative of three experiments.
- FIGURE 4.
Invasion of Matrigel by scp-2 cells expressing activated M-Ras depends on the cell density or the presence of the CM. Parental cells that did not express GFP (1.25 × 105) were mixed with scp-2 cells expressing G22V M-Ras and GFP at either a low density 1.25 × 104 (A and C) or a high density 1.25 × 105 (B) in a final volume of 1 ml and plated on Matrigel in the absence (A and B) or presence (C) of a 25% dilution of 50-fold concentrated medium conditioned by G22V M-Ras scp-2 cells. After 24 h in culture, the bottom of the dish was photographed to show the cells that had invaded through the Matrigel layer. Bar, 20 μm.
- FIGURE 5.
Medium conditioned by scp-2 cells expressing activated M-Ras induced activation of Erk and Akt. G22V M-Ras scp-2 cells (2 × 106) plated in 10 cm dishes were starved for 6 h at 37°C in DMEM followed by stimulation with 10 ml DMEM and 50% of CM from G22V M-Ras scp-2 cells. Other aliquots of cells were treated with TPA or 5% FBS as positive controls or DMSO as a vehicle control. Shown are immunoblots with antibodies recognizing phospho-Erk (A) or phospho-Akt (B). The loading controls were performed by stripping and reblotting the same membrane with antibodies recognizing β-actin.
- FIGURE 6.
Medium conditioned by scp-2 cells expressing G22V M-Ras stimulated the scattering of MDCK cells. As described above, epithelial monolayers of MDCK cells were incubated for 24 h with 1 ml of medium alone (Control) or 1 ml of a 25% dilution of 50-fold concentrated medium conditioned by G22V M-Ras scp-2 cells (CM G22V M-Ras) or GFP scp-2 cells (CM GFP) or 1 ml of a 25% dilution of 50-fold concentrated CM G22V M-Ras that had been premixed with 4 μg/ml antibodies to HGF/SF (anti-HGF). Also shown are parallel cultures in 1 ml medium containing 25 ng recombinant HGF/SF (rHGF) or 1 ml medium containing 25 ng recombinant HGF/SF premixed with 4 μg/ml anti-HGF/SF antibodies. Results shown are representative of three independent experiments. Bar, 40 μm.
- FIGURE 7.
The ability of CM to maintain the cell number or anchorage-independent growth is dependent on the activity of HGF/SF. A. G22V M-Ras scp-2 were cultured at 2.5 × 104/ml in a 5 ml DMEM supplemented with CM G22V M-Ras adjusted to a final concentration of 50% or 5 ml DMEM supplemented with CM G22V M-Ras adjusted to a final concentration of 50% that had been premixed with 5 μg/ml HGF/SF neutralizing antibody. After 4 days, cells were counted. B. Photomicrographs of colonies and the average numbers of colonies/dish grown from G22V M-Ras scp-2 cells that were plated for 14 days in 1 ml DMEM supplemented with 0.3% agar and 5% FBS with CM G22V M-Ras adjusted to 50% or 1 ml of the same medium to which HGF/SF neutralizing antibodies (5 μg/ml) had been added.
- FIGURE 8.
Recombinant HGF/SF is sufficient to maintain number of cells, anchorage-independent growth, and invasion of Matrigel by G22V M-Ras scp-2 cells when plated at low density. A. G22V M-Ras scp-2 cells were cultured for 48 h at low cell density (2.5 × 104/ml) in serum- and insulin-free DMEM in the presence of HGF/SF (25 ng/ml) or PBS as a control. Note the increased numbers of cells in the presence of HGF/SF. B. G22V M-Ras scp-2 cells (103) were plated in 1 ml DMEM supplemented with 0.3% agar and 5% FBS and either PBS, HGF/SF (25 ng/ml), or HGF/SF (25 ng/ml) that had been preincubated with 4 μg/ml anti-HGF/SF antibodies for 30 min. Shown are the colonies after 14 days in culture. C. G22V M-Ras scp-2 cells were plated at low cell density (2 × 103/ml) on Matrigel for 24 h in 1 ml medium supplemented with 4% FBS containing PBS, HGF/SF, or HGF/SF preincubated with 4 μg/ml anti-HGF/SF antibodies. The photomicrographs of the bottom of the dishes show the cells that have invaded through the Matrigel to the bottom of the dish in the cultures supplemented with HGF/SF as well as the spherical mammospheres. D. Prestarved G22V M-Ras scp-2 cells were stimulated for 10 min with HGF/SF (25 ng/ml) or TPA (100 nm) and lysed in lysis buffer. WCLs (25 μl) were subjected to SDS-PAGE and immunoblotting with antibodies specific for activating phosphorylations on Erk. Loading controls were performed by stripping and reblotting of the same membrane with antibodies recognizing β-actin.
- FIGURE 9.
Invasive growth of scp-2 cells expressing activated M-Ras depends on the autocrine factor HGF/SF. G22V M-Ras scp-2 cells (2.5 × 105/ml) were maintained in 0.5% FBS and 5 μg/ml insulin and plated on Matrigel precoated on 35 mm dishes with PBS as control (A) or a final concentration of 10 μg/ml of an antibody neutralizing HGF/SF (B). The photographs were taken after culture for 18 h. Bar, 40 μm. C. After 72 h in culture, the cells were released from Matrigel by digestion with dispase and were counted.
- FIGURE 10.
Expression of activated M-Ras in scp-2 cells results in secretion of MMPs, the activity of which is necessary for invasion of Matrigel. CM from GFP scp-2 or G22V M-Ras scp-2 cells was collected and concentrated 5-fold and CM (10 μl) was subjected to 10% SDS-PAGE under nonreducing conditions in the presence of the substrates described as follows. A. The gel contained 0.1% gelatin and was stained with Coomassie blue to show the electrophoretic mobility of activities that digested gelatin. Also shown is a stained control gel developed in the presence of EDTA to inhibit the activity of gelatinases like MMP-2 and MMP-9. B. The gel contained 0.1% gelatin and plasminogen (20 μg/ml) and was developed in the presence of 10 mm EDTA to inhibit MMPs. Coomassie staining shows an activity that cleaved plasminogen into plasmin that in turn digested the gelatin and was consistent in its Mr with uPA. C. Photomicrographs (40×) of G22V M-Ras scp-2 cells that were cultured for 24 h at 2.5 × 105/ml in Matrigel with the indicated concentrations of a broad-spectrum inhibitor of MMPs, BB94, or with the solvent control DMSO (1%). Shown is dose-dependent inhibition of formation of networks of cell bundles. Also shown are photomicrographs (200×) of control culture or culture treated with 100 nm BB94. Arrows, cells that have invaded the bottom of the dish (control culture). Many fewer invading cells are present in the culture treated with BB94. Bar, 20 μm. Also shown are photomicrographs (40× and 200×) of control GFP scp-2 cells cultured in the presence of DMSO (1%) showing mammospheres.
- FIGURE 11.
Inhibition of Erk activation inhibits invasion of Matrigel by G22V M-Ras scp-2 cells. A. G22V M-Ras scp-2 cells were plated on Matrigel for 24 h at 2.5 × 105/ml in the presence of the indicated concentrations of the MEK-1/2 inhibitor PD98059 or the PI3K inhibitor LY294002 or a solvent control, DMSO (1%). The bottoms of the dishes were photographed after 24 h to show the numbers of cells that had invaded through the Matrigel and had spread out on the bottom of the dish. Bar, 20 μm. B. G22V M-Ras scp-2 cells were cultured for 24 h in the presence of the indicated concentrations of the MEK-1/2 inhibitor PD98059 or DMSO (1%). Shown are immunoblots of SDS-PAGE of cell lysates using antibodies recognizing phospho-Erk or phospho-Akt. The loading controls were performed by stripping and reblotting the same membrane with antibodies recognizing β-actin. C. CM was generated from G22V M-Ras scp-2 cells that had been starved for 6 h and were cultured at 2.5 × 105/ml in DMEM containing PD98059 (12.5 μm; 1), anti-HGF/SF antibodies at a concentration of 20 μg/ml (2) or 10 μg/ml (3), or DMSO as a control (4). A positive control consisting of a supernatant from a cell expressing MMP-9 and MMP-2 was included (5). After 24 h of culture, CM was collected and concentrated 10-fold and the numbers of cells were counted. Volumes of concentrated CM corresponding to 4 × 105 cells were subjected to 10% SDS-PAGE under nonreducing conditions and zymography was performed using 0.1% gelatin as a substrate.
Volume 2, Issue 4
