Involvement of epidermal growth factor receptor in chemically induced mouse bladder tumour progression
Ahmed El Marjou,
Annie Delouvée,
Jean Paul Thiery and
Franciois Radvanyi1
UMR 144, CNRS, Institut Curie, 26 rue d'Ulm, 75248 Paris Cedex 05, France
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Abstract
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This study was designed to investigate the role of the epidermal growth factor receptor (EGFR) and its ligands in chemically induced mouse bladder cancer. Bladder tumours were induced in C57Bl/6 and B6D2F1 mice by treatment with the carcinogen, N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN). The levels of mRNA for EGFR and its ligands were analysed by reverse transcriptionpolymerase chain reaction (RTPCR) in bladder tumours and in normal bladder urothelia. EGFR mRNA was detected in all tumours, transforming growth factor
(TGF
) mRNA levels were similar to those in normal bladder urothelia or were decreased and mRNA levels for amphiregulin, heparin-binding epidermal growth factor-like factor (HB-EGF) and betacellulin were significantly higher than those in normal urothelia. Seven cell lines were derived from chemically induced tumours. These cell lines were able to grow in serum-free conditions. All the cell lines tested expressed the genes encoding EGFR and at least one of its ligands. Proliferation of these cell lines was inhibited by AG1478, a specific EGFR tyrosine kinase inhibitor, strongly suggesting that EGFR was involved in cell growth. As expected, EGFR was found to be phosphorylated in serum-free medium, this phosphorylation being inhibited by AG1478. Conditioned medium of a bladder cancer cell line had EGFR-stimulating activity and an antibody directed against EGFR inhibited proliferation by 45%. This suggests that tumour cell growth is stimulated by an autocrine loop involving EGFR and secreted growth factors. AG1478 decreased the expression of genes for amphiregulin, HB-EGF and betacellulin, showing that EGFR activation induces up-regulation of the EGFR ligands. These results suggest that EGFR plays a critical role in bladder tumour progression.
Abbreviations: BBN, N-butyl-N-(4-hydroxybutyl)nitrosamine; BTC, betacellulin; DMEM, Dulbecco's modified Eagle's medium; EGFR, epidermal growth factor receptor; FCS, fetal calf serum; HB-EGF, heparin-binding epidermal growth factor-like factor; RTPCR, reverse transcriptionpolymerase chain reaction; TBP, TATA-binding protein; TGF
, transforming growth factor
.
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Introduction
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Bladder cancer is the fourth most common neoplasm in men and the ninth most common in women in western countries. Approximately 90% of malignant tumours arising in the urinary bladder are of epithelial origin. Bladder carcinomas comprise two different disease entities. The first, accounting for the majority (80%) of bladder carcinomas, presents as superficial non-muscle invasive papillary lesions. Tumours of this type display a strong tendency to recur but have limited potential to progress to muscle invasion. The second group consists of tumours that are muscle-invasive on presentation, with a much poorer prognosis. For most muscle-invasive tumours, there is no prior history of superficial lesions, and it has been suggested that most invasive tumours are carcinoma in situ derived (1).
The association between specific risk factors and urothelial tumours has been demonstrated by a series of epidemiology studies (2). More than 50% of bladder carcinomas are thought to be associated with cigarette smoking or exposure to industrial carcinogens (35). The carcinogens specifically implicated include aromatic amines and nitrosamines, many of which have been detected in human urine.
Growth factors and their receptors regulate normal cell proliferation and differentiation and may be involved in any of the several steps in neoplastic development and progression. The mechanisms by which growth factor receptors participate in malignant transformation include receptor activation by mutation, autocrine and paracrine growth loops, changes in signalling and regulatory pathways and, possibly, receptor trans-activation (611).
Epidermal growth factor receptor (EGFR), also called ErbB1, was the first identified member of the subfamily of tyrosine kinase receptors that includes ErbB2/Neu, ErbB3 and ErbB4 (12). The ligands of the EGFR belong to the epidermal growth factor (EGF)/transforming growth factor
(TGF
) family, which includes EGF, TGF
, amphiregulin, heparin-binding EGF-like factor (HB-EGF), betacellulin (BTC) and epiregulin (1315). Over-expression of EGFRas a result of gene amplification, increased transcription rate and/or protein stabilizationhas been reported in a wide spectrum of human cancers including non-small cell lung cancer, head and neck squamous carcinomas, glioblastoma multiforme and cancers of the breast, pancreas (9,16,17) and bladder (1820). In several tumour types, including bladder carcinoma, overexpression of EGFR has been shown to be correlated with poor prognosis. Overexpression of EGFR by tumour cells is also often accompanied by the production of one or more of the ligands by the same tumour, suggesting that an autocrine loop may be involved in tumorigenesis (20,21). The effect of EGFR inhibitors on the growth of tumour-derived cell lines supports the hypothesis that EGFR is involved in tumour progression. The use of monoclonal antibodies or specific inhibitors targeting EGFR function may open up new possibilities for effective treatment (22,23).
Highly invasive carcinomas of the urinary bladder are specifically induced in mice by gastric intubation or administration in the drinking water of N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN). This mouse model of chemically induced bladder cancer is invaluable for studying the mechanisms of urinary bladder carcinogenesis, due to its constant genetic background, constant aetiology and well-defined stage of tumour development (2426). The role of growth factors and growth factor receptors has not previously been studied in this model.
In this work, we evaluate the possible involvement of EGFR in this mouse model of bladder cancer. Levels of mRNA for EGFR and its ligands were evaluated in normal urothelium and BBN-induced tumours. The up-regulation of several EGFR ligands together with the presence of the receptor in bladder tumours led us to study the role of EGFR in tumour cell growth, using seven cell lines derived from BBN-induced tumours.
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Materials and methods
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Reagents
BBN was purchased from Tokyo Kasei Kogyo (Tokyo, Japan). Tyrosine kinase inhibitor AG1478 was obtained from France Biochem (Meudon, France) and was dissolved in dimethylsulfoxide to produce a 10 mM stock solution. Anti-EGFR antibody was obtained from Upstate Biotechnology (Lake Placid, NY), anti-phosphotyrosine antibodies (Py20 and 4G10) from Signal Transduction Laboratory (Interchim, Paris, France), anti-rat thymocytes and T cell antibody (IgG2a MRC Ox-34) from Serotech (Oxford, UK) and EGF from Sigma Chemical Co. (Saint-Quentin Fallavier, France). Protein Gagarose, [3H]thymidine (30 Ci/mmol), [
32P]dCTP, the enhanced chemiluminescence (ECL) kit and secondary antibodies were purchased from AmershamPharmacia (Les Ulis, France).
Animals, treatment and tissue samples
Male B6D2F1 and C57BL/6 mice (Iffa Credo, L'Arbresle, France), aged 810 weeks old at the time of the first carcinogen administration, were housed in plastic cages in a controlled-environment room maintained at 22°C ± 1°C with a 12 h light12 h dark cycle. All animals received food and tap water ad libitum.
Animals were given BBN at concentrations of 0.01% or 0.05% (v/v) in their drinking water for 20 weeks (the BBN solution was prepared freshly every 23 days). Mice were killed when they presented symptoms of weakness (between 1 and 10 weeks after the end of the treatment). Part of each tumour was fixed in 10% formalin solution and routinely processed for haematoxylin and eosin staining. The rest of the tumour was immediately frozen in liquid nitrogen and stored at 80°C until used for RNA isolation. Normal bladders were obtained from untreated C57BL/6 (n = 5) and B6D2F1 (n = 5) mice. Urothelium was separated mechanically from the underlying muscle layer, frozen in liquid nitrogen and stored at 80°C until used for RNA isolation.
Cell lines
Seven chemically induced mouse bladder carcinoma cell lines (BC13, BC29, BC30, BC46, BC57, BC58 and BC59) were established in our laboratory from tumours that developed in C57BL/6 mice treated with 0.05% BBN in their drinking water, using the protocol described by Dubeau and Jones (27). The NBT-II cell line, derived from a chemically induced rat bladder carcinoma (28), was obtained from Professor Marc Marel (University Hospital, Gent, Belgium).
BC cell lines were cultured in DMEMF12+ (a 1:1 mixture of Ham's F12 and Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated fetal calf serum (FCS), 1 µg/ml insulin, 2 mM L-glutamine, 10 mM HEPES, 50 nM hydrocortisone, 5 µg/ml apotransferrin, 5 nM sodium selenite, 100 units/ml penicillin and 100 µg/ml streptomycin). The NBT-II cell line was cultured in DMEM supplemented with 10% FCS (29). All cell lines were incubated at 37°C in a humidified atmosphere in 5% CO2.
DMEMF12 supplemented with 10 mM HEPES, 5 µg/ml transferrin, 5 nM sodium selenite, 100 units/ml penicillin and 100 µg/ml streptomycin was used for culture in serum-free medium conditions.
Tritiated thymidine incorporation
Cell proliferation was assayed by measuring the incorporation of [3H]thymidine. Cells were plated at a density of 6000 cells/well in DMEMF12+ and incubated for 24 h to facilitate attachment. They were then washed three times with phosphate-buffered saline containing Ca2+/Mg2+ (PBS Ca2+/Mg2+) and incubated for 48 h in serum-free medium. Growth factors or inhibitors were added and the cells were incubated for a further 2024 h. Cells were then labelled by incubation for 4 h with 1 µCi/well of [3H]thymidine. At the end of the labelling period, the cells were treated with trypsin and transferred to FiltermatA membrane with a Tomtech apparatus (Wallac Inc., Gaithersburg, MD). After several washes, the membrane was dried and the cell-bound radioactivity was determined in a 96-well scintillation counter (Microbeta counter, Wallac Inc.).
Preparation of conditioned medium and effect on cell proliferation
Conditioned medium from BC57 cells was collected and prepared as follows. Cells were cultured in 24 cm2 tissue culture flasks (Falcon) in DMEMF12+. When the cells were 6070% confluent, they were washed twice with PBS Ca2+/Mg2+ and the medium was replaced with serum-free medium. The flasks were incubated for 24 h and the conditioned medium was removed, filtered through a 0.2 µm pore filter and used immediately. The mitogenic activity of the conditioned medium was assessed using a rat bladder carcinoma cell line (NBT-II) in the presence or absence of a specific EGFR inhibitor (AG1478). NBT-II cells were seeded in DMEM containing 10% FCS in 96-well cell culture plates at a density of 2.5 x 104 cells/well. They were incubated until they reached confluence, washed twice with DMEM alone and then incubated for 24 h in serum-free medium. Cells were incubated for a further 2024 h in BC57-conditioned medium at various dilutions in serum-free medium with and without 1 µM AG1478. Proliferation was then assayed by [3H]thymidine incorporation as described above.
RTPCR analysis
Total RNA was extracted by caesium chloride ultracentrifugation (30,31) and used as the template for first-strand cDNA synthesis by random priming, as previously described (32,33). The amount of mRNA was determined by semi-quantitative radioactive RTPCR, using TATA-binding protein (TBP) as an internal control, as previously described (34). The number of cycles was selected so as to be in the exponential part of the PCR reactions. The sequence of the primers, and the sizes of the amplified fragments are given in Table 1
. The PCR-amplified products were subjected to electrophoresis in 8% polyacrylamide gels. Signals were quantified with a Molecular Dynamics 300 PhosphorImager (Molecular Dynamics, Sunnyvale, CA). There was no amplification if reverse transcriptase was omitted from the reverse transcription reaction.
Lysate preparation, western blot analysis and immunoprecipitation
Bladder cancer cell lines were seeded in six-well plates in DMEMF12+. The cells were incubated for 24 h, washed twice with PBS Ca2+/Mg2+, then incubated in serum-free medium. When the cells reached confluence, they were exposed to various concentrations of AG1478 for 16 h. They were then incubated for a further 10 min, with or without 100 ng/ml mouse EGF, and scraped into lysis buffer (50 mM TrisHCl pH 7.4, 1% Nonidet P-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EGTA, 1 mM phenylmethylsulfonylfluoride, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 100 µM sodium orthovanadate, 50 mM NaF). Lysates were kept on ice for 510 min, centrifuged for 10 min at 4°C and 14 000 x g, and the supernatant was collected. Protein concentration was determined with the Bradford protein assay. Lysates were then subjected to western blotting or immunoprecipitation.
For western blot analysis, the proteins (50 µg) in each extract were resolved by 7.5% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE), transferred to PVDF membrane (Immobilon; Millipore, Bedford, MA), and incubated with mouse anti-phosphotyrosine monoclonal antibodies (Py20 and 4G10) at a dilution of 1:5000. Bound antibodies were detected with an anti-mouse horseradish peroxidase-conjugated secondary antibody, using an ECL kit.
For immunoprecipitation analysis, lysates were incubated overnight at 4°C with 12 µg of a sheep polyclonal anti-EGFR antibody. Immunocomplexes were collected by incubation with 30 µl of protein Gagarose for 2 h at 4°C. Immunoprecipitated proteins were washed three times in lysis buffer, resolved by SDSPAGE (7.5%) and immunoblotted with mouse anti-phosphotyrosine monoclonal antibodies. Bound antibodies were detected as described above. To detect EGFR after immunoprecipitation, the blot was stripped of primary and secondary antibodies and reprobed with the sheep polyclonal anti-EGFR antibody. Bound antibody was detected as described above, using an anti-sheep secondary antibody.
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Results
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Expression of EGFR and EGF family members in BBN-induced mouse bladder tumours
Fourteen C57B6 and 26 B6D2 mice were treated for 20 weeks with BBN. At autopsy, all the treated mice presented highly invasive carcinoma of the bladder. Carcinomas exhibited urothelial, squamous or anaplastic differentiation or both urothelial and squamous differentiation. All the tumour samples used in this expression analysis study consisted primarily of tumour cells: samples adjacent to the pieces used for RNA preparation were analysed histologically by haematoxylin/eosin staining and were found to contain >80% tumour cells. Control urothelia were obtained from the bladders of untreated animals by mechanically separating this compartment from the underlying bladder muscle layer. Desmin, a smooth muscle marker, was used to test the quality of the separation. As expected, desmin mRNA was detected exclusively in the muscle layer samples (data not shown).
We assessed the levels of mRNA for EGFR and its ligands, EGF, TGF
, amphiregulin, HB-EGF and BTC, in these bladder tumours and in bladder urothelia from untreated animals (five C57B6 mice and five B6D2 mice). We measured mRNA levels by semi-quantitative RTPCR, using TBP as an internal standard. Two examples of quantification are given in Figure 1A
. Normal urothelia of C57B6 mice contained mainly EGFR and TGF
mRNA whereas amphiregulin, HB-EGF, BTC and EGF mRNA levels were low or barely detectable (Figure 1B
). The EGFR gene was expressed in all bladder tumours and was generally slightly overexpressed compared with normal urothelia (Figure 1A and B
), TGF
mRNA levels were on average similar to those in normal bladder urothelia (Figure 1B
) with some tumours showing a significant down-regulation (data not shown). Levels of amphiregulin, HB-EGF and BTC mRNA were much higher than in normal urothelia (Figure 1A and B
). Very similar results were obtained with mice from a different genetic background (B6D2 mice), exposed to two concentrations of BBN in their drinking water (0.01% and 0.05%) (Figure 1C
), except that the EGF gene was not expressed in any of the tumours.

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Fig. 1. Levels of mRNA for EGFR and its ligands in normal bladder urothelia and in chemically induced mouse bladder tumours. Levels of mRNA for EGFR and its ligands, TGF , amphiregulin (AR), HB-EGF, BTC and EGF, were analysed in BBN-induced bladder tumours and in untreated bladder urothelia (N) by semi-quantitative RTPCR, using TBP as an internal control. (A) Levels of mRNA for EGFR and HB-EGF in normal bladder urothelia and in 14 BBN-induced tumours in C57B/6 mice. The upper band corresponds to the internal control (TBP) and the lower band to the factor analysed. (B) Levels of mRNA for EGFR and its ligands in normal bladder urothelia and in 14 BBN-induced tumours in C57B/6 mice. Tumours were induced by treatment with 0.05% BBN in the drinking water. The reported values are the means for 14 tumours. The bars indicate the standard error. (C) Levels of mRNA for EGFR and its ligands in normal bladder urothelia and in BBN-induced tumours in B6D2 mice. Tumours were induced by treatment with 0.01% or 0.05% BBN in the drinking water. The reported values are the means for 11 tumours treated with 0.01% BBN and 15 tumours treated with 0.05% BBN. The bars indicate the standard error.
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Expression of EGFR and its ligands in cell lines derived from BBN-induced bladder tumours
The presence of EGFR associated with an up-regulation of the ligands HB-EGF, amphiregulin and BTC in BBN-induced tumours suggested that this receptor was involved in autocrine growth in this animal model of bladder carcinoma. To study further the role of EGFR and its ligands in bladder carcinogenesis, we derived seven cell lines from seven C57B6 mouse bladder tumours induced by BBN (BC13, BC29, BC30, BC46, BC57, BC58 and BC59). All these cell lines grew in serum-free conditions, indicating that they were capable of autonomous growth. We then used semi-quantitative RTPCR to evaluate the levels of mRNA for EGFR and its ligands in four of these cell lines cultured in serum-free conditions. All the cell lines tested expressed both EGFR and at least one of its ligands; two cell lines expressed four ligands (Figure 2
) and none of the cell lines expressed EGF (data not shown).

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Fig. 2. Levels of mRNA for EGFR and its ligands in normal bladder urothelia and in mouse bladder cell lines. Levels of mRNA for EGFR and its ligands, TGF , amphiregulin (AR), HB-EGF and BTC, were determined by semi-quantitative PCR in untreated bladder urothelia (N) and in the bladder cell lines, BC29, BC57, BC58 and BC59. These cell lines were derived from BBN-induced tumours. The upper band corresponds to the internal control (TBP) and the lower band to the factor analysed.
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EGFR tyrosine kinase inhibitor decreases the proliferation of mouse bladder cell lines and down-regulates expression of various ligands of EGFR
We investigated the role of EGFR in the autonomous growth displayed by the various cell lines more directly, by studying the effects on proliferation of an EGFR tyrosine kinase-specific inhibitor (AG1478). At a concentration of 1 µM, AG1478 had a marked inhibitory effect (6090%) on thymidine incorporation in the seven bladder cell lines grown in serum-free conditions (Figure 3
). This effect was dose-dependent, with an ID50 of 0.2 µM for the BC57 cell line (data not shown).

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Fig. 3. Effect of the EGFR tyrosine kinase inhibitor, AG1478, on the proliferation of mouse bladder tumour cell lines. Proliferation of the various cell lines derived from BBN-induced tumours was assessed by measuring [3H]thymidine incorporation in the absence () and presence (+) of 1 µM AG1478.
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Several studies have shown that EGFR ligands can induce their own synthesis and that of other members of the EGF family, via activation of EGFR (35). We investigated whether EGFR activation induced the synthesis of EGF family ligands in BC cell lines by determining levels of mRNA for TGF
, amphiregulin, HB-EGF and BTC in BC57 and BC58, in the presence or absence of the EGFR inhibitor, AG1478. BC57 and BC58 were used in this study because they produced these EGFR ligands (Figure 2
). A concentration of 1 µM AG1478 significantly decreased the levels of mRNA for amphiregulin, HB-EGF and BTC in both cell lines, and decreased TGF
mRNA levels in the BC58 cell line only (Figure 4
).

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Fig. 4. Down-regulation of EGFR ligands mRNA by the EGFR tyrosine kinase inhibitor, AG1478. Levels of mRNA for amphiregulin (AR), HB-EGF and BTC were analysed by semi-quantitative RTPCR, using TBP as an internal control, in the cell lines BC57 and BC58, in the absence () and presence (+) of the EGFR inhibitor, AG1478, at a concentration of 1 µM.
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An EGFR-neutralizing antibody inhibited the proliferation of a mouse bladder cell line
We investigated whether EGFR was activated within the cell or at the cell surface. For this purpose, we evaluated the ability of the mouse monoclonal antibody LA22, directed against the extracellular domain of human EGFR, to inhibit thymidine incorporation by the bladder cancer cell line BC57 in serum-free medium. LA22 antibody (50 µg/ml) decreased thymidine incorporation in BC57 cells by 45%, whereas MRC Ox-34, a control antibody of the same isotype (IgG2a) had no effect (Figure 5
).

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Fig. 5. Inhibition by an anti-EGFR antibody of the growth of the mouse bladder cancer cell line BC57 in serum-free medium. The mouse bladder cell line BC57 was grown in serum-free medium in the presence or absence of two different antibodies at two concentrations. L22 is a monoclonal antibody directed against EGFR, whereas OX-34 is a control antibody of the same isotype as L22. The proliferation of BC57 was assessed measuring [3H]thymidine incorporation. The error bars indicate the standard error for eight measurements.
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Conditioned medium of the BC57 bladder cancer cell line contains growth factor activity that can stimulate proliferation of the NBT-II cell line through EGFR
The inhibition by an EGFR antibody of the autonomous growth of BC cell lines suggested that activation of EGFR occurred at the cell surface. This receptor activation may be mediated by a ligand, secreted by the cells, that subsequently binds to EGFR on the cell surface. We therefore investigated whether the conditioned medium of the BC57 cell line contained growth factor activity able to activate EGFR. The NBT-II cell line, which has limited growth in the absence of serum and can respond to EGFR ligands (29), was treated with BC57-conditioned medium in the presence or absence of the EGFR-specific inhibitor AG1478 (Figure 6
). Conditioned medium of the BC57 cell line strongly stimulated proliferation of the NBT-II cell line. This stimulation was completely abolished if the cells were incubated with 1 µM AG1478 together with the conditioned medium.

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Fig. 6. Effect of BC57 conditioned medium on the proliferation of the rat bladder cancer cell line NBT-II in the presence or absence of AG1478. NBT-II cells were treated with the indicated amount of BC57 conditioned medium (CM) alone or in combination with 1 µM AG1478. The proliferation of NBT-II cells was assessed by measuring [3H]thymidine incorporation.
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Inhibition of EGFR autophosphorylation by EGFR tyrosine kinase inhibitor
The probable involvement of EGFR in the autonomous growth of various mouse bladder cell lines led us to investigate the tyrosine phosphorylation of EGFR in the presence and absence of exogenous EGF. In the BC29 cell line, in serum-free medium and in the absence of EGF, a protein of the same molecular weight as EGFR (170 kDa) was detected by western blotting with anti-phosphotyrosine antibodies. As expected, a band of identical molecular weight, but giving a stronger signal, was observed if cells were incubated with exogenous EGF (Figure 7A
). Incubation of BC29 cells with AG1478, a specific EGFR tyrosine kinase inhibitor, resulted in a lower level of phosphorylation of the 170 kDa band. This effect was concentration dependent (Figure 7A
). Immunoprecipitation with an anti-EGFR antibody followed by western blotting with anti-phosphotyrosine antibodies confirmed that the 170 kDa band corresponded to EGFR (Figure 7B
). As expected, no phosphorylation was detected if the cells were treated with AG1478, although EGFR was present in the immunoprecipitates (Figure 7B
). Two phosphorylated proteins of ~100 and 120 kDa were also immunoprecipitated with anti-EGFR antibodies. The phosphorylation of these proteins was inhibited by AG1478. These proteins may be part of the EGFR signal transduction cascade. Similar results were obtained with the BC57 cell line (data not shown).

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Fig. 7. Effect of AG1478 on EGFR tyrosine phosphorylation. (A) Cells from the BC29 cell line grown in the presence or absence of EGF (100 ng/ml) were treated with the indicated concentrations of AG1478. Equivalent amounts of the various cell lysates were subjected to SDSPAGE in 7.5% polyacrylamide gels, and the proteins thus separated were transferred to PVDF membranes and probed with anti-phosphotyrosine monoclonal antibodies. (B) Equivalent amounts of proteins from the BC29 cell line, untreated or treated with 100 ng/ml of EGF in the absence () or presence (+) of 1 µM AG1478, were incubated with an anti-EGFR polyclonal antibody. The immunoprecipitated proteins were subjected to SDSPAGE in 7.5% polyacrylamide gels, transferred to PVDF membranes and probed with an anti-phosphotyrosine monoclonal antibody (upper panel) and with an anti-EGFR polyclonal antibody (lower panel).
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Discussion
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A role for EGFR in oncogenesis has been suggested because a number of human tumours and tumour cell lines have been shown to overproduce EGFR. As TGF
is often overexpressed with EGFR, it has been suggested that this EGFR ligand is an autocrine growth factor (9). In human bladder carcinomas, high EGFR levels have been reported mainly for high-grade and high-stage tumours and have been found to be associated with an increase in the risk of tumour progression (19,36). A limited number of studies performed on human bladder tumour cell lines have shown that EGFR is involved in cell growth (37) and motility (38).
In this study, we provide evidence that EGFR is involved in BBN-induced mouse bladder tumours: (i) the genes encoding EGFR and its ligands, TGF
, amphiregulin, HB-EGF and BTC, were co-expressed in bladder tumours and in derived cell lines; (ii) the levels of mRNA for amphiregulin, HB-EGF and BTC in tumours and derived cell lines were higher than those in normal urothelium; (iii) proliferation of the bladder cancer cell lines was inhibited by AG1478, a tyrosine kinase inhibitor specific for EGFR; (iv) proliferation of the bladder cancer cell lines was inhibited by an antibody directed against the extracellular domain of human EGFR; (v) EGFR was phosphorylated in the cell lines grown in serum-free medium and this phosphorylation was inhibited by treatment of the cells with AG1478; and (vi) the conditioned medium of one of the cell lines (BC57) was found to contain EGFR-stimulating activity, as it increased proliferation of the NBTII cell line and this stimulation was blocked by the EGFR tyrosine kinase inhibitor, AG1478.
EGFR may be activated in tumour cells by several mechanisms that are not mutually exclusive: (i) an increase in the expression of EGFR ligands may be the primary event, which then triggers EGFR activation; (ii) overexpression of EGFR; (iii) abolition of the negative feedback regulation of EGFR (39); (iv) as EGFR ligands are all initially synthesized in a membrane-bound form, proteolytic cleavage to release a soluble mature ligand may be rate-limiting for activation of the receptor; (v) ligand-independent activation of the receptor by trans-activation has also been described recently (40,41). The last four of these mechanisms are not incompatible with the higher levels of ligands observed in mouse bladder tumours and cell lines as ligand overexpression may be induced by EGFR activation, a phenomenon known as autoinduction (35). Consistent with this, we found that the inhibition of EGFR kinase activity by AG1478 considerably reduced overexpression of the various ligands.
EGFR may interact with the EGFR ligands produced by the tumour cells within the cells or at the cell surface. The partial inhibition of EGFR activation by an antibody shows that EGFR activation occurs partly at the cell surface (42,43).
As previously reported, BBN induced invasive bladder tumours of several types in mice (2426). In this study, we observed squamous cell carcinoma, urothelial cell carcinoma, carcinomas that were a mixture of both types and undifferentiated carcinomas. There was no relationship between ligand overexpression and the type of tumour observed (data not shown), suggesting that EGFR is involved in all the types of invasive tumour induced by BBN. In humans, most muscle-invasive tumours overproduce EGFR (44). It would be interesting to study the levels of EGFR and its various ligands in BBN-induced tumours in rats because BBN induces almost exclusively papillary non-muscle-invasive tumours (45).
In human bladder tumours, the expression of genes encoding only two EGFR ligands, EGF and TGF
, has been studied so far (20,21,46). In view of our results in mice, it would be interesting to study other EGFR ligands too.
Our data strongly suggest that EGFR is involved in bladder tumour progression in mice. This model could be used to study in detail the involvement of EGFR in the early stages of tumour progression, which are difficult to study in humans. Metastases are observed in BBN-treated mice (47) so this model could also be used to study late stages of tumour progression. The various ligands of the EGFR are not equivalent in terms of the activation of various pathways from EGFR, ErbB3, ErbB4 dimers or ErbB heterodimers and duration of the activation signal (4851). Mice with these genes knocked out for EGF, TGF
and amphiregulin are available and viable (5254), and the use of such transgenic mice could be used to determine the respective roles played by the various ligands in this bladder tumour model.
Due to the probable role of EGFR in human tumours, intensive studies have been carried out in which EGFR signalling was specifically blocked (22,23). Four potential strategies using EGFR as a therapeutic target are now being studied: (i) monoclonal antibody alone; (ii) immunotoxins; (iii) monoclonal antibodies in conjunction with standard chemotherapy; and (iv) pharmacological agents inhibiting EGFR or downstream components of the EGFR pathway. As EGFR is probably involved in BBN-induced bladder tumour in mice, this animal model could be used to study protocols targeting the EGFR or EGFR pathway. EGFR inhibitors may act at two levels, blocking the activity of the receptor but also decreasing the expression of genes encoding autocrine EGFR ligands.
Evidence has already been obtained to suggest that EGFR is involved in several types of tumour in mice and rats, including skin and oesophageal carcinomas (55,56). Therefore, in mouse models, as in humans, EGFR seems to be involved in several types of malignancy.
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Notes
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1 To whom correspondence should be addressed Email: francois.radvanyi{at}curie.fr 
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Acknowledgments
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We thank Roger Jouffre (SEAT, CNRS, Villejuif, France) for his help with the carcinogenesis experiments, Julie Sappa (Alex Edelman & Associates) for careful reading of the manuscript, and Dominique Chopin for support and fruitful discussions. This work was supported in part by the CNRS and Comité de Paris Ligue Nationale Contre le Cancer (Laboratoire Associé).
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Received August 7, 2000;
accepted September 1, 2000.