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. 1999 Aug;10(8):2493-506.
doi: 10.1091/mbc.10.8.2493.

Radiation-induced release of transforming growth factor alpha activates the epidermal growth factor receptor and mitogen-activated protein kinase pathway in carcinoma cells, leading to increased proliferation and protection from radiation-induced cell death

P Dent  1 , D B Reardon, J S Park, G Bowers, C Logsdon, K Valerie, R Schmidt-Ullrich
Affiliations
Free PMC article

Radiation-induced release of transforming growth factor alpha activates the epidermal growth factor receptor and mitogen-activated protein kinase pathway in carcinoma cells, leading to increased proliferation and protection from radiation-induced cell death

P Dent et al. Mol Biol Cell. 1999 Aug.
Free PMC article

Abstract

Exposure of A431 squamous and MDA-MB-231 mammary carcinoma cells to ionizing radiation has been associated with short transient increases in epidermal growth factor receptor (EGFR) tyrosine phosphorylation and activation of the mitogen-activated protein kinase (MAPK) and c-Jun NH(2)-terminal kinase (JNK) pathways. Irradiation (2 Gy) of A431 and MDA-MB-231 cells caused immediate primary activations (0-10 min) of the EGFR and the MAPK and JNK pathways, which were surprisingly followed by later prolonged secondary activations (90-240 min). Primary and secondary activation of the EGFR was abolished by molecular inhibition of EGFR function. The primary and secondary activation of the MAPK pathway was abolished by molecular inhibition of either EGFR or Ras function. In contrast, molecular inhibition of EGFR function abolished the secondary but not the primary activation of the JNK pathway. Inhibition of tumor necrosis factor alpha receptor function by use of neutralizing monoclonal antibodies blunted primary activation of the JNK pathway. Addition of a neutralizing monoclonal antibody versus transforming growth factor alpha (TGFalpha) had no effect on the primary activation of either the EGFR or the MAPK and JNK pathways after irradiation but abolished the secondary activation of EGFR, MAPK, and JNK. Irradiation of cells increased pro-TGFalpha cleavage 120-180 min after exposure. In agreement with radiation-induced release of a soluble factor, activation of the EGFR and the MAPK and JNK pathways could be induced in nonirradiated cells by the transfer of media from irradiated cells 120 min after irradiation. The ability of the transferred media to cause MAPK and JNK activation was blocked when media were incubated with a neutralizing antibody to TGFalpha. Thus radiation causes primary and secondary activation of the EGFR and the MAPK and JNK pathways in autocrine-regulated carcinoma cells. Secondary activation of the EGFR and the MAPK and JNK pathways is dependent on radiation-induced cleavage and autocrine action of TGFalpha. Neutralization of TGFalpha function by an anti-TGFalpha antibody or inhibition of MAPK function by MEK1/2 inhibitors (PD98059 and U0126) radiosensitized A431 and MDA-MB-231 cells after irradiation in apoptosis, 3-[4, 5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), and clonogenic assays. These data demonstrate that disruption of the TGFalpha-EGFR-MAPK signaling module represents a strategy to decrease carcinoma cell growth and survival after irradiation.

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Figures

Figure 1
Figure 1
Treatment of EGFR-CD533 and EGFR-antisense cells with doxycycline increases expression of EGFR-CD533 and reduces expression of EGFR, respectively. EGFR-CD533 (A) and EGFR-antisense (B) cells were cultured as in MATERIALS AND METHODS and were treated with 1 μg/ml doxycycline for 48 h before experimentation. After 48 h, cells were lysed, and equal total protein amounts (100 μg) were immunoprecipitated, followed by immunoblotting with the same antibody to determine the expression of EGFR and EGFR-CD533. Exposure of A was for 2 min; exposure of B was for 60 s. Representative experiments for EGFR-CD533 (n = 5) and EGFR-antisense (n = 3) cells are shown.
Figure 2
Figure 2
Radiation-induced activation of EGFR in EGFR-antisense cells is blocked by expression of antisense EGFR mRNA. EGFR-antisense cells were cultured and were treated with doxycycline or neutralizing antibody or infected with adenovirus as described in MATERIALS AND METHODS. Cells were irradiated (2 Gy), and the tyrosine phosphorylation of the EGFR was determined over 0–300 min as in MATERIALS AND METHODS. Cells were lysed, and portions (∼100 μg) from each plate were used to immunoprecipitate EGFR, followed by SDS-PAGE and immunoblotting versus either EGFR or phosphotyrosine (active) EGFR (EGFR-phosphotyrosine); a representative experiment is shown; n = 4. Exposure time, 30 s.
Figure 3
Figure 3
Radiation-induced activation of MAPK in EGFR-antisense cells is blocked by expression of antisense EGFR mRNA, tyrphostin AG1478, dominant negative Ras N17, or incubation of cells with a neutralizing antibody to TGFα. EGFR-antisense cells were cultured and were treated with doxycycline or neutralizing antibody or infected with adenovirus as described in MATERIALS AND METHODS. Cells were irradiated (2 Gy), and MAPK activity was determined over 0–300 min as in MATERIALS AND METHODS. Cells were lysed, and portions (∼100 μg) from each plate were used to immunoprecipitate MAPK followed by immune complex kinase assays as in MATERIALS AND METHODS. MAPK activity data are shown as fold increases in 32P incorporation into MBP substrate and are normalized to activity at time = 0 from the means ± SEM of four independent experiments.
Figure 4
Figure 4
Radiation-induced activation of JNK1 is dependent on the functions of TNFR, EGFR, TGFα, and Ras. EGFR-antisense cells were cultured and were treated with doxycycline or neutralizing antibody or infected with adenovirus (A) or treated with neutralizing antibody (B), as described in MATERIALS AND METHODS and the legend to Figure 2. Cells were irradiated (2 Gy), and JNK activity was determined via immunoprecipitation over 0–300 min as in MATERIALS AND METHODS and the legend to Figure 2. JNK activity data are shown as fold increases in 32P-incorporation into GST-c-Jun substrate and are normalized to activity at time = 0 from the means ± SEM of three independent experiments.
Figure 5
Figure 5
Radiation-induced secondary activation of EGFR is dose dependent and is mediated by TGFα. EGFR-antisense cells were cultured and were treated 30 min before irradiation with either control antibody or neutralizing antibody as described in MATERIALS AND METHODS. Cells were exposed to increasing doses of radiation (2–20 Gy). (A and B) After radiation exposure, at the indicated times media were aspirated, and plates were snap frozen; (C) after radiation exposure (180 min), media were aspirated, and plates were snap frozen. Cells were lysed, and identical portions (∼100 μg) from each plate were used to immunoprecipitate EGFR, followed by SDS-PAGE and immunoblotting versus either total EGFR protein or phosphotyrosine-containing (active) EGFR (EGFR-phosphotyrosine) as indicated. A representative experiment is shown (n = 3). Exposure time, 40 s.
Figure 6
Figure 6
Radiation-induced secondary activation of MAPK and JNK is dose dependent and is mediated by TGFα. EGFR-antisense cells were treated 30 min before irradiation with either control antibody (A, MAPK; B, JNK) or anti-TGFα-neutralizing antibody (C, MAPK; D, JNK) as described in MATERIALS AND METHODS. Cells were exposed to increasing doses of radiation (1–20 Gy). At the indicated times after radiation exposure, plates were snap frozen. Cells were lysed, and identical portions (∼100 μg) from each plate were used to immunoprecipitate either MAPK (A and C) or JNK (B and D) for activity assessment as in MATERIALS AND METHODS. Data are shown as fold increases in 32P incorporation into MBP (MAPK) or GST-c-Jun (JNK) substrates and are normalized to activity at time = 0. Data are from a representative of three independent experiments.
Figure 7
Figure 7
Media from irradiated cells contains a soluble factor, which can activate both MAPK and JNK in nonirradiated cells, which is blocked by a neutralizing anti-TGFα antibody. EGFR-CD533 and EGFR-antisense cells (as indicated) were cultured as described in MATERIALS AND METHODS. In D, cells were treated with doxycycline to induce EGFR-CD533 as in MATERIALS AND METHODS. Cells were irradiated (2 Gy), and 0 and 120 min after irradiation the media were removed from these cells. The media were incubated with either neutralizing anti-TGFα antibody or control antibody as in MATERIALS AND METHODS. Antibody-incubated media were added to nonirradiated plates, and 5 min after addition the plates were aspirated and snap frozen. (A) MAPK activity in EGFR-CD533 cells was determined after immunoprecipitation as in MATERIALS AND METHODS; (B) MAPK activity in EGFR-antisense cells was determined after immunoprecipitation as in MATERIALS AND METHODS. (C) JNK activity in EGFR-antisense cells was determined after immunoprecipitation as in MATERIALS AND METHODS. (D) MAPK activity in EGFR-CD533 cells expressing EGFR-CD533 was determined after immunoprecipitation as in MATERIALS AND METHODS. MAPK activity data are shown as fold increases in 32P incorporation into MBP substrate and are normalized to activity at time = 0 from the means ± SEM of three independent experiments. JNK activity data are shown as fold increases in 32P incorporation into GST-c-Jun substrate and are normalized to activity at time = 0 from the means ± SEM of four independent experiments.
Figure 8
Figure 8
Radiation-induced cleavage of 21-kDa pro-TGFα into a 5-kDa active TGFα fragment. EGFR-antisense cells were cultured as described in MATERIALS AND METHODS. Cells were irradiated (2 Gy), and 0, 30, 120, 180, and 300 min after irradiation (as indicated) the media were removed from these cells. Cells were not washed. Cells were immediately lysed in homogenization buffer, followed by immunoprecipitation using an antibody that recognizes both full-length pro-TGFα (∼21.5 kDa) and cleaved active TGFα (∼5 kDa) but not residual proteolytically cleaved TGFα forms (∼17–12 kDa). Proteins were resolved from immunoprecipitates by SDS-PAGE followed by immunoblotting using the same immunoprecipitating antibody, which recognizes pro-TGFα (∼21.5 kDa) and cleaved active TGFα (∼5 kDa), as indicated. A representative experiment is shown (n = 3). Exposure time, 4 min.
Figure 9
Figure 9
Neutralization of TGFα or MAPK function abolishes proliferation after irradiation of EGFR-CD533 cells in a cellular proliferation assay. Decreased proliferative rate of EGFR-CD533 cells in MTT assays over 1–4 d wherein cells were treated with either neutralizing anti-TGFα antibody (2 μg/ml) (A) or the MEK1/2 inhibitor U0126 (2 μM) (B). Cells were cultured in 24-well plates as in MATERIALS AND METHODS. Cells were pretreated with the indicated concentrations of anti-TGFα antibody or U0126 30 min before radiation exposure. Cells were exposed or not exposed to radiation and cultured for 1–7 d in the presence of drug or antibody. MTT assays were performed on each day as described in MATERIALS AND METHODS, and growth curves were plotted to determine the growth potential of treated cells. Data are shown from the means of 12 data points ± SEM from a representative experiment (n = 2).
Figure 10
Figure 10
Inhibition of the MAPK pathway reduces the clonogenic survival of EGFR-antisense cells and EGFR-CD533 cells after irradiation and U0126 treatment. Cells were irradiated (2 Gy) over 3 d (total 6 Gy) in the presence or absence of 2 μM U0126 and DMSO vehicle control. Twenty-four hours after the final irradiation and drug treatment, cells were isolated by trypsinization, and live cell number was determined by hemocytometer. Cells were washed three times in drug-free medium. Their ability to form colonies was determined by a previously described technique. Cells were plated at either 500 or 2500 cells per well. Colonies were counted 14 d after plating, when they contained ≥50 cells. Data are the means of eight separate experiments ± SEM.
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