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First published online May 4, 2004
doi: 10.1242/10.1242/jcs.01057

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Overexpression of the C-terminal PG-M/versican domain impairs growth of tumor cells by intervening in the interaction between epidermal growth factor receptor and ß₁-integrin

Sunnybrook & Women's College Health Sciences Centre and Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON M4N 3M5, Canada

View larger version (65K): [in a new window]   Fig. 1. Expression of G3EGF resulted in cell contact inhibition and reduced cell proliferation. (A) Structures of versican, the versican G3 domain and the G3 domain lacking two EGF-like motifs (G3EGF). (B) Vector- and G3EGF-transfected, and untransfected U87 cells (2x105 cells per well) were cultured on six-well tissue-culture plates to obtain monolayer cultures (–agarose) or on six-well plates coated with 0.6% agarose gel to obtain suspension cultures (+agarose). In the monolayer cultures, the vector-transfected and untransfected cells formed nodules, whereas the G3EGF-transfected cells could only form a monolayer owing to growth contact inhibition. On the agarose-coated plates, the vector-transfected and untransfected cells formed large complexes, whereas the G3EGF-transfected cells formed small conglomerates. (C) Cell growth curves showed that, under monolayer culturing conditions, transfection with G3EGF resulted in a plateau after the cultures reached confluence. (D) On the agarose-coated plates, the vector-transfected cells exhibited higher rate of proliferation than the G3EGF-transfected cells. (E) Expression of G3EGF was confirmed by western blot probed with the monoclonal antibody 4B6. All cell numbers are expressed as the means±SD of three experiments, each performed in triplicate wells,. *P<0.01; **P<0.001.

View larger version (63K): [in a new window]   Fig. 2. Colony formation and tumorigenesis assays. (A) Cells were grown in soft agarose gel. Vector-transfected U87 cells formed colonies but G3EGF-transfected U87 cells did not. Each treatment was done in triplicate wells and the experiment was repeated twice. (B) Both vector- and G3EGF-transfected cells were injected into the nude mice at 107 cells per mouse. Six weeks after injection, tumors were formed by vector-transfected cells (arrows) but not by G3EGF-transfected cells. The photos are results from one experiment. The tumor sizes were measured, and the results from two experiments are given in (C).

View larger version (52K): [in a new window]   Fig. 3. Effects of G3EGF on cell growth under different culture conditions. (A) Cells (2x105 cells per well) were seeded on six-well culture plates maintained in DMEM containing 10% FBS. 1 hour after cell inoculation, G3EGF-transfected U87 cells exhibited spreading on the plates, but vector-transfected U87 cells had only started to attach. Scale bars, 40 µm. (B) Serum was withdrawn from the cultures by changing the culture medium to DMEM alone. At 24 hours, most of the vector-transfected cells had rounded and some had developed long processes to connect to each other, but the G3EGF-transfected cells remained spread on the plates. (C) Cells before and after serum withdrawal were harvested at different time intervals and cell numbers were counted to obtain a growth curve. The total number of the vector-transfected cells declined slowly. However, the G3EGF-transfected cells continued to grow for 5 days, followed by dramatic cell detachment and cell death. (D) Cell death was determined by trypan-blue staining. G3EGF transfection enhanced cell death after serum withdrawal. (E) Cultured in serum-free DMEM, the vector-transfected cells could be maintained for 4 weeks, whereas the numbers of G3EGF-transfected cells declined dramatically owing to cell death. All the experiments were repeated four times. *P<0.01.

View larger version (41K): [in a new window]   Fig. 4. Involvement of the EGFR in G3EGF functions. (A) Cell lysate was prepared from cultures before and after serum withdrawal and immunoprecipitated with anti-EGFR antibody, followed by western blotting probed with anti-phosphotyrosine antibody. U87 cells expressing G3EGF had lower levels of tyrosine-phosphorylated EGFR (pEGFR) after serum withdrawal, but EGFR expression was similar at different time points. Densitometry readings of protein bands are provided below the blots. (B) Both types of cells were starved and then treated with EGF (100 ng/ml) in the presence or absence of AG1478. EGFR phosphorylation was abolished by AG1478 in both types of cells, whereas the protein levels of EGFR were not affected. (C) Cultured in DMEM containing 1% FBS in the presence or absence of AG1478, the growth-inhibitory effect of AG1478 on vector-transfected cells was much more severe than on G3EGF-transfected cells (AG1478– vs AG1478+: **P<0.001; *P<0.05). (D) When AG1478 was added to the cultures after serum withdrawal, cell apoptosis was accelerated more obviously in the vector-transfected cells (from 13% to 45% apoptosis, or a 250% increase) than in G3EGF-transfected cells (from 39% to 52% apoptosis, or a 33% increase). All the experiments were repeated three times.

View larger version (47K): [in a new window]   Fig. 5. Effects of EGF on cell proliferation, apoptosis and EGFR phosphorylation. After serum withdrawal, cultures were maintained in the presence or absence of EGF (40 ng ml–1). Cell numbers were counted at days –1 (cell inoculation in DMEM containing 10% FBS), 0 (serum withdrawal and EGF addition), 1, 3, 5 and 7. The addition of EGF had a greater effect on the growth of G3EGF-transfected U87 cells (A) than vector-transfected U87 cells (B). Data are expressed as the means±SD of three experiments each performed in triplicate. *P<0.01. (C) Cell apoptosis was analysed in the G3EGF-transfected cells. After serum withdrawal, the addition of EGF reduced cell apoptosis to 52% on day 7 compared with cells maintained in the absence of EGF (94% undergoing apoptosis). Cells cultured in DMEM/10% FBS were used as controls for the analyses (n=3). (D) After starvation, the cultures were treated with EGF (100 ng ml–1) for 0, 5 or 20 minutes, as indicated. Cell lysate was prepared for western blotting probed with anti-phosphorylated-EGFR antibody. (Top) 1 minute exposure; (bottom) 30 minutes exposure. (E) The same amount of cell lysate was also analysed and probed with anti-EGFR antibody. G3EGF expression enhanced EGFR turnover (arrow). The same membrane was also probed with anti-actin antibody (arrow) to ensure equal loading of protein samples. Results presented in (D) and (E) were repeated twice.

View larger version (39K): [in a new window]   Fig. 6. G3EGF expression increased and maintained FAK phosphorylation. Cells were cultured in DMEM containing 10% FBS for 24 hours followed by serum withdrawal and incubation in serum-free DMEM for different times. The cell lysate was prepared and immunoprecipitated with anti-FAK antibody, followed by western blotting probed with anti-phosphotyrosine antibody. U87 cells expressing G3EGF retained a prolonged FAK phosphorylation (p-FAK) compared with the vector-transfected cells. The same blot was also probed with anti-FAK antibody to assess equal protein loading. Additionally, the cell lysate was analysed on a western blot probed with anti-ß1-integrin antibody. These experiments were repeated three times.

View larger version (31K): [in a new window]   Fig. 7. G3EGF expression enhanced formation of focal contacts. Cells before and after serum withdrawal were fixed and immunostained with TRITC-labeled phalloidin (which binds to actin filaments) and anti-vinculin antibody. G3EGF-transfected U87 cells formed more and larger focal contacts (stained in yellow) than did vector-transfected U87 cells. After serum withdrawal, some focal contacts could still be detected in G3EGF-transfected cells but not in vector-transfected cells. The experiments were repeated three times and one representative result is shown. Scale bars, 20 µm.

View larger version (38K): [in a new window]   Fig. 8. G3EGF expression promoted EGFR/integrin association. (A) Maintained in different conditions as indicated, U87 cells were lysed and subjected to immunoprecipitation with anti-EGFR antibody, followed by western blot analysis using an anti-ß1-integrin antibody as probe. In the presence of serum, ß1 integrin was precipitated with EGFR. (B) Jurkat cells were cultured in DMEM containing 10% FBS for 4 days. Approximately 20% of the cells attached to the tissue culture plates (adh) and 80% remained in suspension (susp). Both populations of cells were lysed separately with equal amounts of lysis buffer and subjected to immunoprecipitation with anti-EGFR antibody followed by western blot analysis, probed with anti-ß1-integrin antibody. Cell lysate was also analysed by western blotting probed with antibodies against ß1-integrin or EGFR to assess their individual protein levels. Even with lower integrin protein levels (middle), the adherent cultures had higher levels of integrin-EGFR interaction (left). (C) Cell cycles were analysed on the two populations of Jurkat cells. The adherent cells had a larger proportion of cells entering into the S phase (45%) than those in suspension, implying a greater rate of proliferation. (D) G3EGF- and vector-transfected U87 cells were maintained in DMEM containing 10% FBS for 24 hours, followed by serum withdrawal and incubation in serum-free DMEM for different times. Cell lysate was prepared and subjected to immunoprecipitation with anti-EGFR antibody, followed by western blotting probed with anti-ß1-integrin antibody. Expression of G3EGF enhanced the association of EGFR and ß1-integrin before serum withdrawal and delayed the disassociation of EGFR and ß1-integrin after serum withdrawal. Cell lysate was also analysed by western blotting probed with anti-ß1-integrin antibody or anti-EGFR antibody to assess the expression of these two molecules. All experiments were repeated at least three times.