Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2002 Jul;13(7):2233-44.
doi: 10.1091/mbc.01-12-0572.

Epidermal growth factor receptor dependence of radiation-induced transcription factor activation in human breast carcinoma cells

George P Amorino  1 , Virginia M Hamilton, Kristoffer Valerie, Paul Dent, Guido Lammering, Rupert K Schmidt-Ullrich
Affiliations

Epidermal growth factor receptor dependence of radiation-induced transcription factor activation in human breast carcinoma cells

George P Amorino et al. Mol Biol Cell. 2002 Jul.

Abstract

Ionizing radiation (1-5 Gy) activates the epidermal growth factor receptor (EGFR), a major effector of the p42/44 mitogen-activated protein kinase (MAPK) pathway. MAPK and its downstream effector, p90 ribosomal S6 kinase (p90RSK), phosphorylate transcription factors involved in cell proliferation. To establish the role of the EGFR/MAPK pathway in radiation-induced transcription factor activation, MDA-MB-231 human breast carcinoma cells were examined using specific inhibitors of signaling pathways. Gel-shift analysis revealed three different profile groups: 1) transcription factors that responded to both radiation (2 Gy) and epidermal growth factor (EGF) (CREB, Egr, Ets, and Stat3); 2) factors that responded to radiation, but not EGF (C/EBP and Stat1); and 3) those that did not respond significantly to either radiation or EGF (AP-1 and Myc). Within groups 1 and 2, a two- to fivefold maximum stimulation of binding activity was observed at 30-60 min after irradiation. Interestingly, only transcription factors that responded to EGF had radiation responses significantly inhibited by the EGFR tyrosine kinase inhibitor, AG1478; these responses were also abrogated by farnesyltransferase inhibitor (FTI) or PD98059, inhibitors of Ras and MEK1/2, respectively. Moreover, radiation-induced increases in CREB and p90RSK phosphorylation and activation of Stat3 and Egr-1 reporter constructs by radiation were all abolished by AG1478. These data demonstrate a distinct radiation response profile at the transcriptional level that is dependent on enhanced EGFR/Ras/MAPK signaling.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Time-dependent increases in the radiation-induced binding of MDA-MB-231 nuclear extracts to transcription factor consensus sequences. The experimental protocol is described in the legend of Figure 2, except AG1478 treatment was not used in these experiments. Fold-changes represent the mean of three independent experiments.
Figure 2
Figure 2
Inhibition of nuclear extract binding to transcription factor consensus sequences by AG1478 (AG). Cells were preincubated with 5 μM AG1478 for 1 h, irradiated with 2 Gy, and nuclear extracts were isolated at various times. Extracts were incubated with 32P-labeled consensus oligonucleotides, run on TBE-PAGE gels, and autoradiography was performed. The free oligo (FO) samples were incubated without nuclear extract. A 20-fold excess of nonradioactive oligonucleotides were added to the 60 min sample as a “cold” competitor (CC). Fold-changes represent the mean of three independent experiments.
Figure 3
Figure 3
Effect of epidermal growth factor (EGF) treatment on the binding of nuclear extracts to transcription factor consensus sequences. EGF treatments (10 ng/ml) were for 30 min at 37°C before nuclei extraction. For irradiated samples, nuclei were extracted 60 min after a 2-Gy dose. The rest of the protocol is the same as described in the legend of Figure 2. 0, untreated control; R, treatment with radiation alone; E, treatment with EGF alone.
Figure 4
Figure 4
Effect of farnesyltransferase inhibitor I (FTI) on the radiation-induced maximum increase in binding of nuclear extracts to transcription factor consensus sequences. Cells were treated with 50 μM FTI for 18 h before irradiation, and drug was present until protein extraction. Otherwise, the protocol is the same as described in the legend of Figure 2. Fold-changes represent the mean of three independent experiments.
Figure 5
Figure 5
Effect of PD98059 (PD) on the radiation-induced maximum increase in binding of nuclear extracts to transcription factor consensus sequences. Cells were treated with 10 μM PD98059 for 1 h before irradiation, and drug was present until protein extraction. Otherwise, the protocol is the same as described in the legend of Figure 2. Fold-changes represent the mean of three independent experiments.
Figure 6
Figure 6
Gel-shift supershift and cold-competitor controls for transcription factors dependent on the EGFR (CREB, Egr, Ets, and Stat3) or MAPK (C/EBP). Control nuclear extracts (0) were from untreated cells. Supershift antibodies against CREB (all forms), Stat3, Egr-1, Ets-2, C/EBP-α, and C/EBP-β were added to reactions after nuclear extract incubation with the CREB, Stat3, Egr, Ets, and C/EBP oligonucleotides, respectively (SS). A 20-fold excess of nonradioactive oligonucleotides were added to nuclear extracts as a “cold” competitor (CC).
Figure 7
Figure 7
Time-dependent radiation-induced increase in phospho-CREB phosphorylation and inhibition of this effect by AG1478 (1 h pretreatment). Cells were irradiated with 2 Gy, incubated for various times, and extracts were analyzed by Western blotting using phospho-specific antibodies. The bottom panel shows the same blot probed for nonphosphorylated CREB. Fold-changes represent the mean of three independent experiments; maximum fold-increases and inhibition by AG1478 were statistically significant (p < 0.05).
Figure 8
Figure 8
(A) Time-dependent radiation-induced increase in p90RSK phosphorylation. Cells were irradiated with 2 Gy and incubated for various times, and extracts were analyzed by Western blotting. The top panel shows phospho-p90RSK, and the bottom panel shows the same blot probed for β-actin. (B) Inhibition of p90RSK phosphorylation at 30 min after irradiation by either AG1478 or PD98059. Fold-changes represent the mean of three independent experiments. The fold-change at 30 min and inhibition by either compound were significant (p < 0.05).
Figure 9
Figure 9
Radiation-induced increase in reporter construct activation. Cells were cotransfected with reporter constructs containing transcription factor binding sites, and β-galactosidase reporter constructs (to obtain transfection efficiencies values for normalization). Cells were irradiated or EGF-treated and incubated at 37°C for 3 or 6 h. AG1478 was present for 1 h before and after irradiation. Error bars, ±SEM of three independent experiments. Maximum fold-changes and inhibition by AG1478 were statistically significant (p < 0.05). (A) Egr-1 reporter construct; (B) Stat3 reporter construct.
Figure 10
Figure 10
A schematic representation of the effects of EGFR, MAPK, and Ras inhibition on radiation-induced transcription factor activation and proliferation control. The events that are shown downstream of transcription factors in this model are referenced in the DISCUSSION; each transcription factor regulates one or more of the indicated proteins that are involved in proliferation control.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Albanese C, Johnson J, Watanabe G, Eklund N, Vu D, Arnold A, Pestell R. Transforming p21ras mutants and c-Ets-2 activate the cyclin D1 promoter through distinguishable regions. J Biol Chem. 1995;270:23589–23597. - PubMed
    1. Andrisani OM. CREB-mediated transcriptional control. Crit Rev Eukaryot Gene Expr. 1999;9:19–32. - PubMed
    1. Beier F, Lee RJ, Taylor AC, Pestell RG, LuValle P. Identification of the cyclin D1 gene as a target of activating transcription factor 2 in chondrocytes. Proc Natl Acad Sci USA. 1999;96:1433–1438. - PMC - PubMed
    1. Beier F, Taylor AC, LuValle P. The Raf-1/MEK/ERK pathway regulates the expression of the p21(Cip1/Waf1) gene in chondrocytes. J Biol Chem. 1999;274:30273–30279. - PubMed
    1. Beier F, Taylor AC, LuValle P. Activating transcription factor 2 is necessary for maximal activity and serum induction of the cyclin A promoter in chondrocytes. J Biol Chem. 2000;275:12948–12953. - PubMed

Publication types

MeSH terms