Radiotherapy-induced signal transduction

in Endocrine-Related Cancer
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Adly Yacoub
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Anna Miller
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Ruben W Caron
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Liang Qiao
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David A Curiel
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Paul B Fisher
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Michael P Hagan
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Steven Grant
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Paul Dent
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Exposure of tumor cells to ionizing radiation causes compensatory activation of multiple intracellular survival signaling pathways to maintain viability. In human carcinoma cells, radiation exposure caused an initial rapid inhibition of protein tyrosine phosphatase function and the activation of ERBB receptors and downstream signaling pathways. Radiation-induced activation of extracellular regulated kinase (ERK)1/2 promoted the cleavage and release of paracrine ligands in carcinoma cells which caused re-activation of ERBB family receptors and intracellular signaling pathways. Blocking ERBB receptor phosphorylation or ERK1/2 pathway activity using small-molecule inhibitors of kinases for a short period of time following exposure (3 h) surprisingly protected tumor cells from the toxic effects of ionizing radiation. Prolonged exposure (48–72 h) of tumor cells to inhibition of ERBB receptor/ERK1/2 function enhanced radiosensitivity. In addition to ERBB receptor signaling, expression of activated forms of RAS family members and alterations in p53 mutational status are known to regulate radiosensitivity apparently independent of ERBB receptor function; however, changes in RAS or p53 mutational status, in isogenic HCT116 cells, were also noted to modulate the expression of ERBB receptors and ERBB receptor paracrine ligands. These alterations in receptor and ligand expression correlated with changes in the ability of HCT116 cells to activate ERK1/2 and AKT after irradiation, and to survive radiation exposure. Collectively, our data in multiple human carcinoma cell lines argues that tumor cells are dynamic and rapidly adapt to any single therapeutic challenge, for example, radiation and/or genetic manipulation e.g. loss of activated RAS function, to maintain tumor cell growth and viability.

Abstract

Exposure of tumor cells to ionizing radiation causes compensatory activation of multiple intracellular survival signaling pathways to maintain viability. In human carcinoma cells, radiation exposure caused an initial rapid inhibition of protein tyrosine phosphatase function and the activation of ERBB receptors and downstream signaling pathways. Radiation-induced activation of extracellular regulated kinase (ERK)1/2 promoted the cleavage and release of paracrine ligands in carcinoma cells which caused re-activation of ERBB family receptors and intracellular signaling pathways. Blocking ERBB receptor phosphorylation or ERK1/2 pathway activity using small-molecule inhibitors of kinases for a short period of time following exposure (3 h) surprisingly protected tumor cells from the toxic effects of ionizing radiation. Prolonged exposure (48–72 h) of tumor cells to inhibition of ERBB receptor/ERK1/2 function enhanced radiosensitivity. In addition to ERBB receptor signaling, expression of activated forms of RAS family members and alterations in p53 mutational status are known to regulate radiosensitivity apparently independent of ERBB receptor function; however, changes in RAS or p53 mutational status, in isogenic HCT116 cells, were also noted to modulate the expression of ERBB receptors and ERBB receptor paracrine ligands. These alterations in receptor and ligand expression correlated with changes in the ability of HCT116 cells to activate ERK1/2 and AKT after irradiation, and to survive radiation exposure. Collectively, our data in multiple human carcinoma cell lines argues that tumor cells are dynamic and rapidly adapt to any single therapeutic challenge, for example, radiation and/or genetic manipulation e.g. loss of activated RAS function, to maintain tumor cell growth and viability.

Introduction

Ionizing radiation is used as a primary treatment for many types of carcinoma. While it has been appreciated for many years that radiation causes cell death, it has only recently become accepted that radiation has some potential to enhance proliferation in the surviving fraction of cells (Schmidt-Ullrich et al. 2000, Grant et al. 2002a,b). We and other researchers have discovered that exposure of carcinoma cells to low radiation doses causes activation of growth factor receptors in the plasma membrane (Goldkorn et al. 1997, Dent et al. 1999). Receptor activation enhances the activities of RAS family molecules that signal to cause activation of multiple intracellular signal transduction pathways.

Growth factors interact with plasma membrane receptors, which transduce signals through the membrane to its inner leaflet (Sizemore et al. 1999, Fanton et al. 2001, Ludde et al. 2001, Moriuchi et al. 2001, Pruitt & Der 2001, Balmanno et al. 2003, Blalock et al. 2003, Cox & Der 2003). Growth factor signals, via guanine nucleotide exchange factors, can increase the amount of GTP bound to membrane-associated GTP-binding proteins, including RAS (Sklar 1988, Pruitt & Der 2001). There are three widely recognized isoforms of RAS: Harvey (H), Kirsten (K), and Neuroblastoma (N) (Reuther & Der 2000). GTP–RAS can interact with multiple downstream effector molecules including the Raf-1 protein kinase and the phosphatidylinositol 3-kinase (PI3K) lipid kinase. Receptor-stimulated guanine nucleotide exchange of ‘RAS’ to the GTP-bound form permits Raf-1 and P110 PI3K to associate with ‘RAS’, resulting in kinase translocation to the plasma membrane environment where activation of these kinases, via complex mechanisms, takes place. RAS contains a GTPase activity that converts bound GTP to GDP, resulting in inactivation of the RAS molecule. PI3K enzymes are also translocated to the plasma membrane environment via the P85 SH2-domain interaction with phosphorylated tyrosine residues on adaptor proteins and growth factor receptors, e.g. GAB2, IRS-1, and ERBB3 (Hellyer et al. 2001).

Mutation of RAS results in a loss of GTPase activity, generating a constitutively active RAS molecule that can lead to elevated activity within downstream signaling pathways. Approximately, one third of human cancers have RAS mutations, primarily the K-RAS isoform that also leads to a radioprotected phenotype (Sklar 1988, Ellis & Clark 2000). Of note is that some studies suggest that K-RAS and H-RAS have different but over-lapping signaling specificities to downstream pathways as judged by in vitro cell-based studies and in animal knock-out models; thus mutant K-RAS is thought to preferentially activate the Raf-1/extracellular regulated kinase (ERK1/2) pathway, whereas mutant H-RAS is believed to preferentially activate the PI3K/AKT pathway (Yan et al. 1998, Liebmann 2001, Ross et al. 2001). It has been argued that ERK1/2 and PI3K signaling downstream of K-RAS and H-RAS respectively, can in turn control cell growth and cell survival following exposure to multiple growth factors, e.g. Epidermal growth factor (EGF) (Dent et al. 1999, Ludde et al. 2001, Moriuchi et al. 2001).

Loss of the single allele of mutated active K-RAS expression has been shown in HCT116 cells to abolish tumor formation in athymic mice and enhance radio-sensitivity (Baba et al. 2000, Ries et al. 2000). The findings presented in these studies were linked to reduced expression of the paracrine growth factor epiregulin. Repeated irradiation of tumor cells can also increase expression of transforming growth factor α (TGFα) and ERBB1 (Schmidt-Ullrich et al. 1994). Increased proliferative rates and poor prognosis of carcinomas in vivo have also been correlated with increased expression of ERBB1 (Putz et al. 1999).

The studies described herein examine the role of p53, ERBB receptors, ERBB receptor paracrine ligands, and mutated active RAS proteins in the signaling and survival responses of multiple human carcinoma cell lines exposed to ionizing radiation. Radiation exposure causes inhibition of protein tyrosine phosphatase function and the activation of ERBB receptors and downstream signaling pathways, for example, ERK1/2 and AKT. Activation of ERK1/2 promoted the cleavage and release of paracrine ligands, which caused re-activation of ERBB family receptors and intracellular signaling pathways. Expression of activated forms of RAS family members and alterations in p53 mutational status were noted to regulate radiosensitivity in isogenic cells, in part, by modulating the expression of ERBB receptors and ERBB receptor paracrine ligands. These alterations in receptor and ligand expression correlated with changes in the ability of HCT116 cells to activate ERK1/2 and AKT after irradiation, and survive radiation exposure. Thus, tumor cells are dynamic and rapidly adapt to any single therapeutic challenge to maintain tumor cell growth and viability.

Materials and methods

Materials

Anti-phospho-ERK1/2, anti-Raf-1, anti-total phosphortyrosine, anti-total-ERK2, anti-β-actin antibodies, and protein A/G-conjugated agarose were from Santa Cruz Biotechnology (Santa Cruz). Anti-phospho-Ser473-AKT, anti-phospho-Thr308-AKT, anti-total-AKT, anti-phospho-Ser259-RAF, anti-phospho-Tyr1289 ERBB3, anti-phospho-Tyr1173 ERBB1, and anti-phospho-Tyr1187 ERBB2 antibodies were from Cell Signaling Technology (Beverly, MA, USA). Antibodies to detect total RAS, H-RAS and mutant K-RAS, and the neutralizing anti-heregulin antibody and the neutralizing anti-TGFα antibody were from Oncogene Research Products (Cambridge, MA, USA). Antibodies to detect ERBB1, ERBB2, ERBB3, and ERBB4 and for immunoprecipitation of ERBB3 were from Neomarkers Lab Vision Corporation (Fremont, CA, USA). Antibodies for anti-PI3K p85 and anti-PI3K p110α were from Upstate Biotechnology (Lake Placid, NY, USA). Oligonucleotides for semi-quantitative PCR were synthesized by the VCU oligonucleotide core facility and were based on published sequences. Reverse transcriptase-PCR (RT-PCR) was performed using a commercial kit (Quiagen OneStep RT-PCR) and 1 μg RNA in a final volume of 25 μl and following the instructions of the manufacturer (Qiagen). The MEK1/2 inhibitors PD98059, U0126, and PD184352, the PI3K inhibitor LY294002, the ERBB1 inhibitor AG1478, the ERBB2 inhibitor AG825 were from Calbiochem (San Diego, CA, USA; Caron et al. 2005a,b).

Methods

Generation of HCT116 cell lines without mutant K-RAS and expressing mutant H-RAS

HCT116 mutant K-RAS-deleted cells were generated by homologous deletion of the mutant K-RAS allele as described (Shirasawa et al. 1993, Baba et al. 2000, Caron et al. 2005a,b). A plasmid to express mutant active H-RAS (H-RAS V12) was kindly provided by the laboratory of Dr M Wigler (Cold Spring Harbor, NY, USA). HCT116 cell lines were transfected by electroporation at 600 V for 60 ms using a Multi-porator Eppendorff (Hamburg, Germany) with control plasmids (‘C2’ cells) or plasmids to express H-RAS V12 (‘C10’ and ‘C3’ cells). Pools of transfected cells were obtained by puromycin (RAS) selection and individual colonies isolated and then characterized.

Culture of HCT116 cell lines

Asynchronous HCT116 carcinoma cells were cultured in DMEM media supplemented with 10% (v/v) fetal calf serum at 37 °C in 95% (v/v) air/5% (v/v) CO2. Cells were plated at a density 3×103 cells/cm2 plate area and all cells were plated from log-phase cultures. For radiation-induced activations of protein kinases, cells were cultured for 4 days in this media, and 24 h prior to irradiation, were cultured in serum-free DMEM medium. For colony formation assays, cells were plated at low density (250–2000 cells per dish), and 24 h after plating, for the 24 h prior to irradiation, they were cultured in serum-free DMEM medium. Cells were irradiated (1–4 Gy) or as indicated in the text; media was replaced with serum-containing media 24 h after radiation exposure. Plates were washed in PBS 10–14 days after exposure, fixed with methanol, and stained with a filtered solution of crystal violet (5% w/v). After washing with tap water, the colonies were counted both manually (by eye) and digitally using a ColCount TM plate reader. Data presented are the arithmetic mean (± s.e.m.) from both counting methods from multiple studies.

Culture of A431, DU145, and MDA-MB-231 cells

Cells were cultured in RPMI-1640 medium supplemented with 5% (v/v) fetal calf serum at 37 °C in 95% (v/v) air/5% (v/v) CO2. For radiation-induced activation of kinases, cells were cultured for 4 days and for 24 h prior to irradiation were cultured in serum-free medium.

Exposure of cells to ionizing radiation and cell homogenization

Cells were cultured as described above. As indicated, 1 h prior to irradiation, cells were treated with either vehicle (DMSO), U1026 (1 μM), PD184352 (2 μM), or LY294002 (1 μM). Treatment was from a 100 mM stock solution and the maximal concentration of vehicle (DMSO) in media was 0.01% (v/v). Cells were irradiated using a 60Co source at dose rate of 1.8 Gy/min. Cells were maintained at 37 °C throughout the experiment, except during the irradiation itself. Zero time is designated as the time point at which exposure to radiation ceased. After radiation-treatment, cells were incubated for specified times followed by aspiration of media and immediately homogenized in either lysis buffer for immune complex kinase or phosphatase assays (see below), or 1 ml SDS-PAGE lysis buffer (5% (w/v) SDS, 40% (v/v) glycerol, 250 mM Tris–HCl, and 10% (v/v) 2-mercaptoethanol). Homogenates were sonicated, boiled for 10 min, the protein concentration determined by Bradford assay (Coomassie Protein Assay Kit, Pierce Biotechnology, Rockford, IL, USA), and stored frozen (−20 °C) prior to use.

Immunoprecipitation from cell lysates

After irradiation, cells were incubated for specified times, followed by aspiration of media and snap-freezing at 70 °C on dry ice. Cells were lysed and Raf-1 and ERK1/2 activity determined as described by Dent et al. (1995a,b).

Protein tyrosine phosphatase assay

Cellular PTP activity was assessed by an in vitro assay with autophosphorylated EGFR as substrate (Dent et al. 2003a,b). EGFR was purified from A431 cells by affinity chromatography on lentil lectin Sepharose as previously described (Tomic et al. 1995).

TGFα ELISA/concentration of release assay

ELISA kits for mammalian TGFα were obtained from Oncogene. Assays were performed according to the manufacturer’s protocol with slight modifications. Briefly, cells were grown to 60% confluence in RPMI medium alone or supplemented with AG1478, PD98059, 60 min before exposure to 60Co-radiation, and the media sampled after various intervals of incubation at 37 °C. Medium sample and TGFα standard in triplicate were added to the TGFα-pre-coated wells and incubated for 4 h at room temperature. Samples were then processed according to the manufacturer’s protocols. Media without cells, and media incubated with EGF were used as a negative control in each assay. All reactions were measured using a spectrophotometric plate reader at a wavelength of 490 nm (BioTek Instruments, Inc., Highland Park, VT, USA).

SDS-PAGE and western blotting

Depending on the protein to be studied, a volume of homogenate containing 10, 20, or 40 μg total protein was loaded in 12% (w/v) acrylamide gels and subjected to SDS-PAGE. Gels were transferred to nitrocellulose and western blotted using specific antibodies. Blots were developed using Western Lightning Chemiluminescence Reagent Plus (Perkin–Elmer Life Sciences, Boston, MA, USA) and Kodak X-ray film, Densitometric analysis for ECL immunoblots, and RT-PCR analyses was performed using a Fluorochem 8800 Image System and software (Alpha Innotech Corporation, San Leandro, CA, USA) and band densities were normalized to that of β-actin in the same sample and expressed as a percentage of the respective control in each experiment as indicated in the figure legend.

Cell death assays: Wright Giemsa for apoptosis

Cells were plated at 5×104 cells per well in 12-well plates and 24 h later, serum-starved. The plates were mock-exposed or irradiated 24 h later at 1 or 4 Gy and harvested 96 h after irradiation by trypsinization followed by centrifugation onto glass slides (cytospin) at 800 r.p.m. for 10 min. The cells were fixed and stained with a commercial kit (Diff-Quik) following the instruction of the manufacturer (Dade Behring AG, Düdingen, Switzerland). Randomly selected fields of stained cells (~200 cells per field, n=5 per slide) were counted for apoptotic nuclear morphology.

Membrane preparation from HCT116 cells.

Cells were cultured as described above. Cells were scraped into 10 ml 1 mM NaHCO3 pH 7.4, 1 mM Na pyrophosphate, 1 mM Na orthovanadate, 1 mM EDTA, 1 mM EGTA, 0.1 mM PMSF, and incubated for 1 h at 4 °C, 24 h after serum withdrawal/starvation. Crude membrane preparations were prepared as described by Dent et al. (1995a,b) by sucrose density over-layer centrifugation.

Statistical analysis

Comparison of the effects of treatments was performed using one-way ANOVA and a two-tailed t-test. Differences with a P value <0.05 were considered statistically significant. Experiments shown, except where indicated, are the means of multiple individual points from multiple separate experiments (± s.e.m.).

Results and discussion

Exposure of A431 human carcinoma cells to ionizing radiation (2 Gy) caused inactivation of total cellular protein tyrosine phosphatase (PTPase) activity that correlated with enhanced phosphorylation of ERBB1 (Fig. 1A). Incubation of cells with the reactive oxygen species (ROS) quenching agent N-acetyl cysteine suppressed the reduction in PTPase activity and suppressed the activation of ERBB1. These findings argue that radiation can induce ROS to promote activation of plasma membrane receptors (Leach et al. 2001, Sturla et al. 2005). Downstream of growth factor receptor signaling are RAS family signal transducers and proximal to these proteins in the ERK1/2 pathway are the Raf family protein kinases (Dent et al. 1995a,b). Radiation activated Raf-1, but not B-Raf, in A431 cells in a manner that was also suppressed by incubation of cells with N-acetyl cysteine (Fig. 1B). Of note, Raf-1 becomes tyrosine phosphorylated after radiation exposure, whereas B-Raf lacks the sites of Raf-1 tyrosine phosphorylation (Y340/Y341) (Fig. 1B, inset).

Raf-1 has been proposed to be negatively regulated by phosphorylation at S259, putatively catalyzed by AKT; thus it is possible that dephosphorylation of Raf-1 (S259) could also contribute to enzyme activation following radiation exposure (Reusch et al. 2001). In wild-type HCT116 cells that express active K-RAS D13, in the C2 HCT116 line that has been deleted for the K-RAS D13 allele, and in HCT116 cells deleted for K-RAS D13 expression that are stably transfected to express active H-RAS V12 (termed C10), we noted that basal levels of Raf-1 S259 phosphorylation were elevated > 10-fold in the C10 cell line (Fig. 1C, inset). However, basal levels of phospho-ERK1/2 and phospho-AKT were near identical in both cell lines (Fig. 1C, left-hand side of graphs). Furthermore, in wild-type, but not C10 cells, the majority of Raf-1 protein was membrane associated, whereas in C10, but not wild-type cells, the majority of AKT was membrane associated, that is, membrane location of Raf-1 and AKT also did not correlate with phosphorylation of Raf-1 S259 (Fig. 1C, left-hand side of graphs). Radiation promoted similar-fold reductions in Raf-1 S259 phosphorylation in both cell lines, which again did not correlate well with downstream alterations in ERK1/2 activity in both lines (Fig. 1C, graphical keys). Activation of ERK1/2 followed a similar pattern in A431 cells after irradiation to that noted in HCT116 cells (Figs 1C and 2A). Collectively, these findings argue that ROS play an important role in the activation of ERBB1 and Raf-1 after ionizing radiation exposure, and that Raf-1 tyrosine phosphorylation but not Raf-1 S259 phosphorylation correlates well with the activation of a key downstream target of Raf-1 signaling, the MEK1/2–ERK1/2 pathway.

As noted in Fig. 1C for HCT116 carcinoma cells, radiation also appeared to cause two waves of ERK1/2 activation in A431 carcinoma cells; an initial prompt wave that occurred within 5–15 min exposure and a second delayed wave that occurred 90–360 min after exposure (Fig. 2A). In A431 carcinoma cells, the activation of ERBB1 and ERK1/2 5–15 and 90–360 min after exposure was blocked by the tyrphostin inhibitor of ERBB1, AG1478 (Fig. 2A). In A431 cells, neutralization of the paracrine factor-(TGFα) suppressed the activation of ERBB1 60–180 min after a 2 Gy exposure (Fig. 2B, section (i)). In HCT116 cells deleted for K-RAS D13 expression, which are stably transfected to express active H-RAS V12 (C10), neutralization of the paracrine factor heregulin suppressed the activation of ERBB3 60–180 min after a 1 Gy exposure (Fig. 2B, section (ii)). Thus, the second wave of ERBB receptor activation after irradiation of carcinoma cells is due to the actions of paracrine ligands; in a cell-type dependent fashion, multiple ligands can play a role in this process. In general agreement with our findings at the level of the ERBB receptors, incubation of tumor cells with neutralizing antibodies against TGFα or against heregulin also suppressed the second, but not the first wave, of radiation-induced signaling pathway activation, either ERK1/2 or AKT as shown in the Figure, in multiple carcinoma cell lines (Fig. 2C).

Paracrine factors are synthesized as pro-forms that are cleaved by cell surface metalloproteases; as such, we predicted that media from irradiated cells should contain cleaved/activated TGFα or heregulin in a cell-type dependent fashion, and that could be transferred onto unirradiated cells to induce signaling pathway activation. In agreement with this hypothesis, media transferred from irradiated HCT116 (C10) cells promoted heregulin-dependent activation of AKT in unirradiated HCT116 (C10) cells (Fig. 2D). Furthermore, treatment of HCT116 cells in situ with a neutralizing anti-heregulin antibody also suppressed AKT activation in the irradiated cells (compare with data in Fig. 2C). Similarly, in A431 cells, media transferred from these irradiated carcinoma cells promoted TGFα-dependent activation of ERK1/2 in unirradiated A431 cells (Fig. 2D). In A431 and DU145 cells, radiation promoted TGFα release into the growth media, as measured in an ELISA assay (Fig. 2E, left panel). Release of paracrine factors, e.g. TGFα, was also blocked by an inhibitor of MEK1/2, implying that the initial activation of ERK1/2 signaling promoted paracrine ligand release, of note, the initial ERK1/2 activation was ROS-dependent and paracrine ligand-independent. Incubation of cancer cells with an inhibitor of cell surface metalloproteases e.g. GM6001, abolished the ability of media from irradiated tumor cells to promote AKT activation in non-irradiated cells, suggesting that radiation-induced release of a paracrine ligand had been blocked (Fig. 2E, right panel). Collectively, our data in Figs 1 and 2 demonstrate that radiation generates ROS, which inactivates PTPases that permits activation of ERBB receptors, and which activates ERK1/2 and AKT signaling, all within 30 min of exposure (Fig. 2F). The levels of ROS and pathway activities then subside. Subsequently, radiation-induced ERK1/2 signaling causes activation of cell surface metalloproteases which in turn cleave and activate pro-forms of paracrine ligands. The activated paracrine ligands then feedback onto the irradiated carcinoma cell to re-activate ERBB receptors and the ERK1/2 and AKT signaling pathways, 90–360 min after exposure. Of importance for therapeutics is that radiation-induced TGFα release has not only been observed using cell lines, in vitro and in vivo, but has also been observed in androgen-independent prostate cancer patients undergoing palliative irradiation for bony metastases (Hagan et al. 2004). Thus, inhibition of ERBB1 and/or MEK1/2 may prevent radiation-induced TGFα release in patients; TGFα release being an effect that is likely to promote the growth and invasion of unirradiated tumor cells at sites distant to the radiation exposure (Schmidt-Ullrich et al. 1999).

Based on the findings in Figs 1 and 2, it could be simplistically presumed that inhibiting ERBB1 and/or ERK1/2 activation would result in tumor cell radiosensitization. However, while continuous exposure of A431 and DU145 cells for 24 h to MEK1/2 inhibitor did modestly enhance the ability of radiation to promote apoptosis, removal of the drug 6 h after irradiation, abolished the potentiation of apoptosis at 24 h (Fig. 3A). Furthermore, removal of the MEK1/2 inhibitor 24 and 48 h after irradiation abolished the ability of MEK1/2 inhibition to promote cell killing 105 h after exposure in DU145 cells (Fig. 3B, data not shown). These findings argue that for the inhibition of MEK1/2–ERK1/2 signaling to have any radiosensitizing effect in carcinoma cells requires prolonged pathway inhibition. Based on the findings in Fig. 3A and B, colony formation analyses were performed to examine the impact of ERBB1 and MEK1/2 inhibitors on A431, DU145, and MDA-MB-231 radiosensitivity. Inhibition of ERBB1 or MEK1/2 signaling for the 3 h during and after irradiation had the potential to cause radioprotection, that is, ERK1/2 signaling was toxic to cells (Fig. 3C). Removal of the MEK1/2 inhibitor 48 h after exposure had null effect on cell survival, whereas prolonged exposure to MEK1/2 inhibitor promoted radiosensitization when cells were continually cultured in the drug. Collectively, these findings argue that the duration of kinase inhibitor exposure upon a carcinoma cell can dramatically alter the impact on cell survival after radiation exposure.

In general, inhibition of PI3K–AKT signaling has been proposed to generate a stronger radioprotective signal than that generated from MEK1/2–ERK1/2 (Dent et al. 2003a,b). To investigate these phenomena, we examined ERBB receptor, paracrine ligand, and ERK1/2 and AKT signaling in isogenic HCT116 cells. Wild-type HCT116 cells express a single allele of K-RAS D13, the C2 HCT116 line has been deleted for the K-RAS D13 allele, the C10 and C3 clones of HCT116 cells represent clones of C2 stably transfected to express low levels of H-RAS V12, and p53−/−HCT116 cells are wild-type HCT116 cells expressing K-RAS D13 and deleted for both alleles of wild-type p53 (Fig. 4A, right RT-PCR and immunoblotting panels).

Loss of K-RAS D13 expression reduced the levels of the paracrine ligand epiregulin in HCT116 (C2) cells, but enhanced expression of the paracrine ligand heregulin. Of greater note, loss of K-RAS D13 expression enhanced the levels of ERBB1 and ERBB3 expressed in the C2 and C10 HCT116 cell lines, suggesting that the tumor cell, on losing the function of K-RAS D13 adapted to its environment/loss of RAS and epiregulin function, to survive by increasing the expression of ERBB1 and ERBB3, and heregulin. Of additional note, loss of p53 function reduced ERBB1 expression by approximately 80% and enhanced expression of ERBB2 and ERBB3 compared with wild-type cells (Fig. 4A, left bar graph). The increased expression of ERBB2 and ERBB3 in p53−/− cells may thus also be a compensatory survival response of cells deleted for p53 expression. No change in the expression of RAS proteins or paracrine ligands was observed comparing HCT116 wild-type with HCT116 p53−/− (Fig. 4A, right PCR data, data not shown). Hence, although HCT116 cells (wild-type; C2; C10; p53−/−) are scientifically defined as being ‘isogenic’, our data demonstrate that these cells display considerably different expression profiles of signaling molecules that in other carcinoma cell types can impact on how the tumor cell may subsequently respond to irradiation.

In general agreement with our data in Fig. 4A, the activation of ERK1/2 and AKT following radiation exposure was noted to be very different in wild-type, C2, C10, and p53−/− HCT116 cell lines. In concurrence with several studies examining H-RAS and K-RAS signaling in general, cells expressing K-RAS D13 generated a stronger ERK1/2 activation after irradiation than cells expressing H-RAS V12, whereas cells expressing H-RAS V12 generated a stronger AKT activation after irradiation than cells expressing K-RAS D13 (Fig. 4B and C). Of additional note, in cells lacking p53 expression, basal levels of phospho-ERK1/2 and phospho-AKT were reduced and the activation of ERK1/2 and AKT following radiation exposure was suppressed (Fig. 4B and C; phospho-ERK1/2 reduced to 0.75±0.05; phospho-AKT reduced to 0.89±0.05, of control). The findings with respect to K-RAS and H-RAS activation correlated with wild-type, but not C10, HCT116 cells having Raf-1 translocated to their plasma membrane and C10, but not wild-type, cells having PI3K and AKT associated with their plasma membrane (Fig. 1C). Thus, by translocating either Raf-1 (via K-RAS D13) or PI3K and AKT (via H-RAS V12), it is probable that we have predisposed our wild-type and C10 HCT116 cells to preferentially activate ERK1/2 or AKT respectively.

Thus, in ‘isogenic’ HCT116 cells in Figs 2–4 argue that wild-type HCT116 cells expressing epiregulin, K-RAS D13, and low levels of ERBB1-3, promote a stronger activation of ERK1/2 than AKT after irradiation, whereas in HCT116 (C10) cells expressing heregulin, H-RAS V12 and higher levels of ERBB1 and ERBB3, promote a stronger activation of AKT than ERK1/2 after irradiation. In HCT116 cells lacking p53 function, an approximately 80% reduction in ERBB1 expression correlated with reduced activation of ERK1/2 and AKT following irradiation, suggesting that loss of p53 function may have the potential to either enhance radiosensitivity via loss of AKT and ERK1/2 activation or also promote radioresistance by loss of p53-induced apoptosis after DNA damage.

To further investigate the relative roles of ERBB1 and ERBB2 in HCT116 cell signaling after radiation exposure, cells were incubated with the ERBB1 inhibitor AG1478 or with the ERBB2 inhibitor AG825. Inhibition of ERBB1, but not ERBB2, suppressed radiation-induced tyrosine phosphorylation of ERBB1, ERBB2, and ERBB3 in HCT116 (C10) cells (Fig. 5A). Similarly, incubation with AG1478 abolished radiation-induced activation of ERK1/2 and AKT in HCT116 (C10) cells (Fig. 5B). Our findings in HCT116 (C10) cells are in general agreement with data obtained in other human carcinoma cell lines e.g. wild-type HCT116, A431, MDA-MB-231, and DU145 with respect to ERBB1 activation being a central player in radiation-induced ERK1/2 and AKT signaling responses (Fig. 5C).

Based on studies in Figs 4 and 5, changes in RAS family member activity and p53 activity would be predicted to alter cellular plating efficiency and radio-sensitivity in in vitro colony formation assays. C10 HCT116 cells, which express H-RAS V12 and exhibit strong radiation-induced AKT activation, were noted to have a greater plating efficiency and be more radio-resistant than wild-type HCT116 cells, which express K-RAS D13 and exhibit strong radiation-induced ERK1/2 activation; both cell lines were more radio-resistant than HCT116 cells lacking expression of any mutated RAS protein (Fig. 6A). Loss of p53 function, in our colony assays, surprisingly resulted in a finding that p53−/− HCT116 cells were modestly more radiosensitive than wild-type HCT116 cells; an effect that we presume is due to a loss of radiation-induced ERK1/2 and AKT activation in this cell line (Fig. 6B). Thus, loss of p53 function in HCT116 cells, which would be presumed to be radioprotective is outweighed in the overall survival response by loss of pro-survival AKT and ERK1/2 signaling. HCT116 wild-type cells, in agreement with this line exhibiting a stronger activation of ERK1/2 than AKT after irradiation, were radio-sensitized by a MEK1/2 inhibitor (Fig. 6C). HCT116 (C10) cells, in agreement with this line exhibiting a stronger activation of AKT than ERK1/2 after irradiation, were radiosensitized by a PI3K inhibitor. In HCT116 (C2) cells, radiosensitization by signaling pathway interruption was achieved only when both MEK1/2 and PI3K were inhibited. This would tend to argue that inhibition of both PI3K and MEK1/2 may best achieve radiosensitization in the widest range of tumor cell types, regardless of RAS protein expression.

Collectively, our data demonstrate that tumor cells are highly dynamic with respect to the impact of proto-oncogenes on stress-induced changes in cellular signaling, and, as such, tumor cells are noted to rapidly adapt to any single therapeutic challenge e.g. radiation or loss of activated RAS/p53 function, to maintain tumor cell growth and viability. This adaptation can result in the elevated expression of growth-factor receptors e.g. ERBB3 and paracrine ligands, which activate such receptors e.g. heregulin. Therapeutic intervention to fully compromise the long-term survival of a tumor cell will thus require intervention at multiple levels (paracrine ligand, receptor, transducer, and kinase cascade) and within multiple pathways (several ligands, several receptors, several transducers, and multiple kinase cascades) to achieve a therapeutic effect.

Figure 1
Figure 1

Ionizing radiation generates ROS in human carcinoma cells, which inhibit PTPases leading to activation of ERBB1 and Raf-1. (A) A431 cells were plated and serum starved for 24 h prior to pre-treatment for 30 min with 20 mM N-acetyl cysteine or PBS vehicle. Cells were irradiated (2 Gy). In the lower panel, cells were isolated at the indicated time points after exposure and PTPase activity measured in cell lysates. In the upper immunoblotting panel, cells were isolated, ERBB1 immunoprecipitated, and total ERBB1 phospho-tyrosine levels measured (n=3, a representative experiment). (B) A431 cells were plated and serum starved for 24 h prior to pre-treatment for 30 min with 20 mM N-acetylcysteine or PBS vehicle. Cells were irradiated (2 Gy). In the lower panel, cells were isolated at the indicated time points after exposure, Raf-1 or B-Raf immunoprecipitated, and Raf-1 and B-Raf activity measured in vitro against MEK1. Data shown are the fold change in 32P-incorporation into MEK1. In the upper panel, the phospho-tyrosine content of immunoprecipitated Raf-1 was measured (n=3, a representative experiment). (C) HCT116 cells (wild-type, mutant K-RAS deleted termed C2, and C2 cells expressing H-RAS V12, termed C10) were plated and serum starved for 24 h prior to irradiation (1 Gy). Cells were isolated at the indicated time points and immunoblotted after SDS-PAGE to determine total expression of ERK2 and β-actin (loading) as well as the phosphorylation status of Raf-1 (S259); ERK1/2, AKT T308; AKT S473. Graphical data on the left represents Raf-1 S259 and ERK1/2 phosphorylation normalized to total ERK2 protein levels (± s.e.m., n=3). For studies examining membrane-associated proteins on the right, HCT116 wild-type (WT), (C2) and expressing H-RAS V12 (C10) cells were plated in parallel and serum starved for 24 h. Cells were then lysed with hypotonic buffer prior to preparation of plasma membranes. Equal amounts of membrane protein were loaded onto SDS-PAGE and immunoblotting performed against the indicated membrane-associated proteins. A representative experiment is presented (n=3–4).

Citation: Endocrine-Related Cancer Endocr Relat Cancer 13, Supplement_1; 10.1677/erc.1.01271

Figure 2
Figure 2
Figure 2

The actions of paracrine ligands are essential for the re-activation of ERBB family receptors 60–360 min after an initial radiation exposure. (A) A431 cells were plated and serum starved for 24 h prior to pre-treatment for 30 min with 100 nM AG1478 or DMSO vehicle (VEH). Cells were irradiated (2 Gy). In the lower panel, cells were isolated at the indicated time points after exposure, ERK1/2 immunoprecipitated and ERK1/2 activity measured against myelin basic protein in vitros.e.m., n=3). In the upper immunoblotting panel, cells were isolated, ERBB1 immunoprecipitated and total ERBB1 phospho-tyrosine levels measured. (B) A431 (upper section (i)) or HCT116 (C10) cells (lower section (ii)) were plated and serum starved for 24 h prior to pre-treatment for 30 min with either a control IgG, neutralizing anti-TGFα IgG, or a neutralizing anti-heregulin IgG, as indicated. Cells were irradiated; A431 (2 Gy); HCT116 (C10) (1 Gy). In the upper panel (i), cells were isolated, ERBB1 immunoprecipitated, and total ERBB1 phospho-tyrosine levels measured. In the lower panel (ii), cells were isolated, total protein separated by SDS-PAGE, and total and phospho-ERBB3 immunoblotted (n=3, a representative study). (C) Human carcinoma cells were plated and serum starved for 24 h prior to irradiation with 2 Gy (A431, DU145, MDA-MB-231) or 1 Gy (HCT116 (C10)). For 2 Gy irradiated cells, cells were isolated at the indicated time points after exposure, ERK1/2 immunoprecipitated, and ERK1/2 activity measured against myelin basic protein in vitros.e.m., n=3). For 1 Gy irradiated cells, cells were isolated, total protein separated by SDS-PAGE, and total and phospho-AKT (S473) levels immunoblotted (± s.e.m., n=3–5). (D) HCT116 (C10) and A431 cells were plated and serum starved for 24 h prior to irradiation. For HCT116 (C10) with antibody in situ treatment, cells were pre-treated for 30 min with either a control IgG or a neutralizing anti-heregulin IgG, as indicated. For all studies, cells were irradiated; A431 (2 Gy); HCT116 (C10) (1 Gy). In the upper panel: for antibody in situ, cells were isolated, total protein separated by SDS-PAGE, and total ERK2 and phospho-AKT (T308) immunoblotted. For media transfer, media was removed from the irradiated or mock-irradiated plates, incubated with either a control IgG or a neutralizing anti-heregulin IgG, as indicated, and added to plates of unirradiated HCT116 (C10) cells. Cells were isolated, total protein separated by SDS-PAGE, and total ERK2 and phospho-AKT (T308) immunoblotted (n=3, a representative experiment). In the lower panel: for media transfer in A431 cells, media was removed from the irradiated or mock-irradiated plates, incubated with either a control IgG or a neutralizing anti-TGFα IgG, as indicated, and added to plates of the respective unirradiated A431 cells. Media treated cells were isolated, ERK1/2 immunoprecipitated and ERK1/2 activity measured against myelin basic protein in vitros.e.m., n=3). (E) Left panel: A431 and DU145 cells were plated and serum starved for 24 h prior to pre-treatment for 30 min with 10 μM PD98059 or DMSO vehicle (VEH). Cells were irradiated (2 Gy). Media was removed 2 h after exposure and the release of TGFα into the medium determined by ELISA assay (± s.e.m., n=3). Right panel: HCT116 (C10) cells were plated and serum starved for 24 h prior to pre-treatment for 30 min with 10 μM GM6001 or DMSO vehicle (VEH). Cells were irradiated (1 Gy). Media was removed 3 h after exposure from the irradiated or mock-irradiated plates and added to plates of unirradiated HCT116 (C10) cells. Cells were isolated, total protein separated by SDS-PAGE, and total ERK2 and phospho-AKT (T308) immunoblotted; data presented are the fold increase in AKT phosphorylation (± s.e.m., n=4). (F) Putative mechanism by which radiation causes activation of intracellular signaling pathways; generation of ROS leads to activation of receptor tyrosine kinases, then downstream signaling pathways, which in turn, causes cleavage and activation of paracrine ligands that re-activate the entire signaling system.

Citation: Endocrine-Related Cancer Endocr Relat Cancer 13, Supplement_1; 10.1677/erc.1.01271

Figure 3
Figure 3

The duration of MEK1/2 and ERBB1 inhibition can alter the impact of radiation exposure on cell survival; protection, no effect, sensitization. (A) A431 and DU145 cells were plated and 24 h after plating treated with vehicle (VEH, DMSO) or 10 μM PD98059 (PD). After 30 min, cells were irradiated (2 Gy). Media was removed and replaced with either media-containing vehicle or PD98059, 6 h after irradiation. Cell viability (apoptosis, TUNEL assay) was measured on isolated cells 24 h after exposure (± s.e.m., n=3). (B) DU145 cells were plated and 24 h after plating treated with vehicle (VEH, DMSO) or 10 μM PD98059 (PD). After 30 min, cells were irradiated (7.5 Gy). Media was removed and replaced with either media containing vehicle or with PD98059, 24 h after irradiation. Cell viability (apoptosis, TUNEL assay) was measured on isolated cells 105 h after exposure (± s.e.m., n=3). (C) MDA-MB-231, A431, and DU145 cells were plated as single cells and 14 h after plating, treated with vehicle control (DMSO), AG1478 (1 μM) or PD98059 (10 μM) as indicated. Cells were irradiated 30 min after kinase inhibitor addition (2 Gy). At the indicated time after irradiation, media was removed from the assays and replaced by media that did not contain kinase inhibitors. Colonies were permitted to form over the following 10–14 days (± s.e.m., n=3). (*P<0.05 less survival than corresponding irradiated cell value; P<0.05 the greater survival than corresponding irradiated cell value).

Citation: Endocrine-Related Cancer Endocr Relat Cancer 13, Supplement_1; 10.1677/erc.1.01271

Figure 4
Figure 4

Deletion of K-RAS D13 expression or p53 wild-type expression causes multiple changes in the ERBB receptor expression and ERBB paracrine ligand expression profile of HCT116 cells. (A) Right upper section: levels of mRNA for H-RAS, K-RAS, and N-RAS in HCT116 cell clones. The levels of H-, K-, N-RAS, and β-actin were determined by RT-PCR. The data shows quantitative RT-PCR H-RAS, K-RAS, and N-RAS mRNA levels from a representative experiment showing data from WT, C2 (mutant K-RAS deleted), C10 (mutant K-RAS deleted expressing H-RAS V12), and C3 (mutant K-RAS deleted expressing H-RAS V12). Right lower section: heregulin protein expression by western blot and heregulin expression by mRNA content in WT, C2, and C10 cells. Left graphical section: expression of ERBB1, ERBB2, and ERBB3 proteins determined by western blot using receptor-specific antibodies. Densitometry values were normalized with respect to the total amount of β-actin in each sample and then expressed as a percentage with the value for ERBB1 expression in wild-type cells being defined as a 100% value. Bars represent mean ± s.e.m. n=4. (*P<0.05 greater than corresponding WT cell value; P<0.05 less than corresponding WT cell value). Expression of ERBB4 was not detected in HCT116 cells (data not shown). (B) Alteration of ERK1/2 phosphorylation 0–6 h after 1 Gy exposure in HCT116 cells (WT), HCT116 p53−/−, mutant K-RAS-deleted cells (C2) or mutant K-RAS-deleted cells expressing mutant H-RAS V12 (C10). Densitometry values were normalized with respect to total β-actin protein expression and are expressed as a percentage of ERK1/2 phosphorylation in WT cells at t=0. Bars represent mean ± s.e.m., n=3. (C) Alteration of AKT S473 phosphorylation 0–6 h after a 1 Gy exposure in WT, C2, and C10 cells. Densitometry values were normalized with respect to total β-actin protein expression and expressed as a percentage of AKT phosphorylation in WT cells at t=0. Bars represent mean ± s.e.m., n=4.

Citation: Endocrine-Related Cancer Endocr Relat Cancer 13, Supplement_1; 10.1677/erc.1.01271

Figure 5
Figure 5

Inhibition of ERBB1, but not ERBB2, suppresses radiation-induced activation of ERBB1, ERBB2, and ERBB3 in HCT116 (C10) cells. (A) HCT116 (C10) cells were plated, serum starved for 24 h, and treated with either a vehicle control (VEH, DMSO), AG1478 (1 μM), or AG825 (5 μM), 30 min prior to irradiation (1 Gy). Cells were isolated 0–180 min as indicated after exposure and the phosphorylation of ERBB1, ERBB2, and ERBB3 determined after SDS-PAGE and immunoblotting. A representative experiment (n=3). (B) Alteration of ERK1/2 and AKT (S473) phosphorylation 0–6 h after a 1 Gy exposure in HCT116 cells (C10) treated with (VEH, DMSO), AG1478 (1 μm) or AG825 (5 μm), 30 min prior to irradiation (1 Gy). Densitometry values were normalized with respect to β-actin expression and are expressed as a percentage of ERK1/2 or AKT phosphorylation in WT cells at t=0. Bars represent mean ± s.e.m. from three experiments. (C) MDA-MB-231, A431, HCT116 (WT), and DU145 cells were plated and 24 h afterwards treated with vehicle (VEH, DMSO) or with AG1478 (1 μM) followed by exposure to radiation (2 Gy). Cells were lysed and ERK1/2 immunoprecipitated and ERK1/2 activity measured in vitro against myelin basic protein, 5 min after exposure. Data shown are the fold change in 32P-incorporation into MBP (± s.e.m., n=3–4).

Citation: Endocrine-Related Cancer Endocr Relat Cancer 13, Supplement_1; 10.1677/erc.1.01271

Figure 6
Figure 6

Expression of H-RAS V12 causes a greater radio-protective/survival effect than K-RAS D13 in HCT116 cells. (A) The plating efficiency (colonies formed per total number of cells plated) of HCT116 cells (WT), mutant K-RAS-deleted cells (C2) or mutant K-RAS-deleted cells expressing mutant H-RAS V12 (C10, C3) and the survival of these cells when exposed to ionizing radiation (0–4 Gy). Single HCT116 cells were plated and 4 h after plating serum-starved for 24 h before exposure to the respective dose of radiation, media-containing serum added 24 h after exposure and 10–14 days later, colonies were counted as indicated in Materials and methods. The relative efficiency of cell plating (percentage colonies recovered per total number of cells plated) was calculated making the plating efficiency value for WT=1.00. The numbers of colonies were then expressed as a fraction of the respective mock-irradiated cells and plotted in a semi-log graph. Data are the means of six separate dishes from three separate experiments ± s.e.m. (*P<0.05 greater than corresponding WT cell value; P<0.05 less than corresponding WT cell value; ‡P<0.001 greater than mutant K-RAS-deleted cells value). (B) Identical plating efficiency and colony formation studies to those described in (A) were performed using wild-type HCT116 cells, HCT116 (C2) cells and HCT116 p53−/−cells. (C) HCT116 cells were plated and treated with U0126 or LY294002 as indicated, 24 h after plating. Cells were irradiated (5 Gy). Cells were re-plated as single cells for colony formation assays, 96 h after irradiation. Colonies were permitted to form over the following 10 days. The numbers of colonies were expressed asa fraction of the respective mock-irradiated cells and plotted in a semi-log graph. Data are the means of six separate dishes (s.e.m., n=3) (*P<0.05 less than corresponding vehicle treated cell value; P<0.05 less than corresponding LY294002 treated cell value).

Citation: Endocrine-Related Cancer Endocr Relat Cancer 13, Supplement_1; 10.1677/erc.1.01271

This work was funded to PD from PHS grants (R01-CA88906, P01-CA72955, R01-DK52825, P01-CA104177, R01-CA108520), Department of Defense Awards (BC980148, BC020338) to SG from PHS grants (P01-CA72955; R01-CA63753; R01-CA77141), and a Leukemia Society of America grant 6405-97. PD is the holder of the Universal, Inc., Professorship in Signal Transduction Research. The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.

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  • Ionizing radiation generates ROS in human carcinoma cells, which inhibit PTPases leading to activation of ERBB1 and Raf-1. (A) A431 cells were plated and serum starved for 24 h prior to pre-treatment for 30 min with 20 mM N-acetyl cysteine or PBS vehicle. Cells were irradiated (2 Gy). In the lower panel, cells were isolated at the indicated time points after exposure and PTPase activity measured in cell lysates. In the upper immunoblotting panel, cells were isolated, ERBB1 immunoprecipitated, and total ERBB1 phospho-tyrosine levels measured (n=3, a representative experiment). (B) A431 cells were plated and serum starved for 24 h prior to pre-treatment for 30 min with 20 mM N-acetylcysteine or PBS vehicle. Cells were irradiated (2 Gy). In the lower panel, cells were isolated at the indicated time points after exposure, Raf-1 or B-Raf immunoprecipitated, and Raf-1 and B-Raf activity measured in vitro against MEK1. Data shown are the fold change in 32P-incorporation into MEK1. In the upper panel, the phospho-tyrosine content of immunoprecipitated Raf-1 was measured (n=3, a representative experiment). (C) HCT116 cells (wild-type, mutant K-RAS deleted termed C2, and C2 cells expressing H-RAS V12, termed C10) were plated and serum starved for 24 h prior to irradiation (1 Gy). Cells were isolated at the indicated time points and immunoblotted after SDS-PAGE to determine total expression of ERK2 and β-actin (loading) as well as the phosphorylation status of Raf-1 (S259); ERK1/2, AKT T308; AKT S473. Graphical data on the left represents Raf-1 S259 and ERK1/2 phosphorylation normalized to total ERK2 protein levels (± s.e.m., n=3). For studies examining membrane-associated proteins on the right, HCT116 wild-type (WT), (C2) and expressing H-RAS V12 (C10) cells were plated in parallel and serum starved for 24 h. Cells were then lysed with hypotonic buffer prior to preparation of plasma membranes. Equal amounts of membrane protein were loaded onto SDS-PAGE and immunoblotting performed against the indicated membrane-associated proteins. A representative experiment is presented (n=3–4).

  • The actions of paracrine ligands are essential for the re-activation of ERBB family receptors 60–360 min after an initial radiation exposure. (A) A431 cells were plated and serum starved for 24 h prior to pre-treatment for 30 min with 100 nM AG1478 or DMSO vehicle (VEH). Cells were irradiated (2 Gy). In the lower panel, cells were isolated at the indicated time points after exposure, ERK1/2 immunoprecipitated and ERK1/2 activity measured against myelin basic protein in vitros.e.m., n=3). In the upper immunoblotting panel, cells were isolated, ERBB1 immunoprecipitated and total ERBB1 phospho-tyrosine levels measured. (B) A431 (upper section (i)) or HCT116 (C10) cells (lower section (ii)) were plated and serum starved for 24 h prior to pre-treatment for 30 min with either a control IgG, neutralizing anti-TGFα IgG, or a neutralizing anti-heregulin IgG, as indicated. Cells were irradiated; A431 (2 Gy); HCT116 (C10) (1 Gy). In the upper panel (i), cells were isolated, ERBB1 immunoprecipitated, and total ERBB1 phospho-tyrosine levels measured. In the lower panel (ii), cells were isolated, total protein separated by SDS-PAGE, and total and phospho-ERBB3 immunoblotted (n=3, a representative study). (C) Human carcinoma cells were plated and serum starved for 24 h prior to irradiation with 2 Gy (A431, DU145, MDA-MB-231) or 1 Gy (HCT116 (C10)). For 2 Gy irradiated cells, cells were isolated at the indicated time points after exposure, ERK1/2 immunoprecipitated, and ERK1/2 activity measured against myelin basic protein in vitros.e.m., n=3). For 1 Gy irradiated cells, cells were isolated, total protein separated by SDS-PAGE, and total and phospho-AKT (S473) levels immunoblotted (± s.e.m., n=3–5). (D) HCT116 (C10) and A431 cells were plated and serum starved for 24 h prior to irradiation. For HCT116 (C10) with antibody in situ treatment, cells were pre-treated for 30 min with either a control IgG or a neutralizing anti-heregulin IgG, as indicated. For all studies, cells were irradiated; A431 (2 Gy); HCT116 (C10) (1 Gy). In the upper panel: for antibody in situ, cells were isolated, total protein separated by SDS-PAGE, and total ERK2 and phospho-AKT (T308) immunoblotted. For media transfer, media was removed from the irradiated or mock-irradiated plates, incubated with either a control IgG or a neutralizing anti-heregulin IgG, as indicated, and added to plates of unirradiated HCT116 (C10) cells. Cells were isolated, total protein separated by SDS-PAGE, and total ERK2 and phospho-AKT (T308) immunoblotted (n=3, a representative experiment). In the lower panel: for media transfer in A431 cells, media was removed from the irradiated or mock-irradiated plates, incubated with either a control IgG or a neutralizing anti-TGFα IgG, as indicated, and added to plates of the respective unirradiated A431 cells. Media treated cells were isolated, ERK1/2 immunoprecipitated and ERK1/2 activity measured against myelin basic protein in vitros.e.m., n=3). (E) Left panel: A431 and DU145 cells were plated and serum starved for 24 h prior to pre-treatment for 30 min with 10 μM PD98059 or DMSO vehicle (VEH). Cells were irradiated (2 Gy). Media was removed 2 h after exposure and the release of TGFα into the medium determined by ELISA assay (± s.e.m., n=3). Right panel: HCT116 (C10) cells were plated and serum starved for 24 h prior to pre-treatment for 30 min with 10 μM GM6001 or DMSO vehicle (VEH). Cells were irradiated (1 Gy). Media was removed 3 h after exposure from the irradiated or mock-irradiated plates and added to plates of unirradiated HCT116 (C10) cells. Cells were isolated, total protein separated by SDS-PAGE, and total ERK2 and phospho-AKT (T308) immunoblotted; data presented are the fold increase in AKT phosphorylation (± s.e.m., n=4). (F) Putative mechanism by which radiation causes activation of intracellular signaling pathways; generation of ROS leads to activation of receptor tyrosine kinases, then downstream signaling pathways, which in turn, causes cleavage and activation of paracrine ligands that re-activate the entire signaling system.

  • The duration of MEK1/2 and ERBB1 inhibition can alter the impact of radiation exposure on cell survival; protection, no effect, sensitization. (A) A431 and DU145 cells were plated and 24 h after plating treated with vehicle (VEH, DMSO) or 10 μM PD98059 (PD). After 30 min, cells were irradiated (2 Gy). Media was removed and replaced with either media-containing vehicle or PD98059, 6 h after irradiation. Cell viability (apoptosis, TUNEL assay) was measured on isolated cells 24 h after exposure (± s.e.m., n=3). (B) DU145 cells were plated and 24 h after plating treated with vehicle (VEH, DMSO) or 10 μM PD98059 (PD). After 30 min, cells were irradiated (7.5 Gy). Media was removed and replaced with either media containing vehicle or with PD98059, 24 h after irradiation. Cell viability (apoptosis, TUNEL assay) was measured on isolated cells 105 h after exposure (± s.e.m., n=3). (C) MDA-MB-231, A431, and DU145 cells were plated as single cells and 14 h after plating, treated with vehicle control (DMSO), AG1478 (1 μM) or PD98059 (10 μM) as indicated. Cells were irradiated 30 min after kinase inhibitor addition (2 Gy). At the indicated time after irradiation, media was removed from the assays and replaced by media that did not contain kinase inhibitors. Colonies were permitted to form over the following 10–14 days (± s.e.m., n=3). (*P<0.05 less survival than corresponding irradiated cell value; P<0.05 the greater survival than corresponding irradiated cell value).

  • Deletion of K-RAS D13 expression or p53 wild-type expression causes multiple changes in the ERBB receptor expression and ERBB paracrine ligand expression profile of HCT116 cells. (A) Right upper section: levels of mRNA for H-RAS, K-RAS, and N-RAS in HCT116 cell clones. The levels of H-, K-, N-RAS, and β-actin were determined by RT-PCR. The data shows quantitative RT-PCR H-RAS, K-RAS, and N-RAS mRNA levels from a representative experiment showing data from WT, C2 (mutant K-RAS deleted), C10 (mutant K-RAS deleted expressing H-RAS V12), and C3 (mutant K-RAS deleted expressing H-RAS V12). Right lower section: heregulin protein expression by western blot and heregulin expression by mRNA content in WT, C2, and C10 cells. Left graphical section: expression of ERBB1, ERBB2, and ERBB3 proteins determined by western blot using receptor-specific antibodies. Densitometry values were normalized with respect to the total amount of β-actin in each sample and then expressed as a percentage with the value for ERBB1 expression in wild-type cells being defined as a 100% value. Bars represent mean ± s.e.m. n=4. (*P<0.05 greater than corresponding WT cell value; P<0.05 less than corresponding WT cell value). Expression of ERBB4 was not detected in HCT116 cells (data not shown). (B) Alteration of ERK1/2 phosphorylation 0–6 h after 1 Gy exposure in HCT116 cells (WT), HCT116 p53−/−, mutant K-RAS-deleted cells (C2) or mutant K-RAS-deleted cells expressing mutant H-RAS V12 (C10). Densitometry values were normalized with respect to total β-actin protein expression and are expressed as a percentage of ERK1/2 phosphorylation in WT cells at t=0. Bars represent mean ± s.e.m., n=3. (C) Alteration of AKT S473 phosphorylation 0–6 h after a 1 Gy exposure in WT, C2, and C10 cells. Densitometry values were normalized with respect to total β-actin protein expression and expressed as a percentage of AKT phosphorylation in WT cells at t=0. Bars represent mean ± s.e.m., n=4.

  • Inhibition of ERBB1, but not ERBB2, suppresses radiation-induced activation of ERBB1, ERBB2, and ERBB3 in HCT116 (C10) cells. (A) HCT116 (C10) cells were plated, serum starved for 24 h, and treated with either a vehicle control (VEH, DMSO), AG1478 (1 μM), or AG825 (5 μM), 30 min prior to irradiation (1 Gy). Cells were isolated 0–180 min as indicated after exposure and the phosphorylation of ERBB1, ERBB2, and ERBB3 determined after SDS-PAGE and immunoblotting. A representative experiment (n=3). (B) Alteration of ERK1/2 and AKT (S473) phosphorylation 0–6 h after a 1 Gy exposure in HCT116 cells (C10) treated with (VEH, DMSO), AG1478 (1 μm) or AG825 (5 μm), 30 min prior to irradiation (1 Gy). Densitometry values were normalized with respect to β-actin expression and are expressed as a percentage of ERK1/2 or AKT phosphorylation in WT cells at t=0. Bars represent mean ± s.e.m. from three experiments. (C) MDA-MB-231, A431, HCT116 (WT), and DU145 cells were plated and 24 h afterwards treated with vehicle (VEH, DMSO) or with AG1478 (1 μM) followed by exposure to radiation (2 Gy). Cells were lysed and ERK1/2 immunoprecipitated and ERK1/2 activity measured in vitro against myelin basic protein, 5 min after exposure. Data shown are the fold change in 32P-incorporation into MBP (± s.e.m., n=3–4).

  • Expression of H-RAS V12 causes a greater radio-protective/survival effect than K-RAS D13 in HCT116 cells. (A) The plating efficiency (colonies formed per total number of cells plated) of HCT116 cells (WT), mutant K-RAS-deleted cells (C2) or mutant K-RAS-deleted cells expressing mutant H-RAS V12 (C10, C3) and the survival of these cells when exposed to ionizing radiation (0–4 Gy). Single HCT116 cells were plated and 4 h after plating serum-starved for 24 h before exposure to the respective dose of radiation, media-containing serum added 24 h after exposure and 10–14 days later, colonies were counted as indicated in Materials and methods. The relative efficiency of cell plating (percentage colonies recovered per total number of cells plated) was calculated making the plating efficiency value for WT=1.00. The numbers of colonies were then expressed as a fraction of the respective mock-irradiated cells and plotted in a semi-log graph. Data are the means of six separate dishes from three separate experiments ± s.e.m. (*P<0.05 greater than corresponding WT cell value; P<0.05 less than corresponding WT cell value; ‡P<0.001 greater than mutant K-RAS-deleted cells value). (B) Identical plating efficiency and colony formation studies to those described in (A) were performed using wild-type HCT116 cells, HCT116 (C2) cells and HCT116 p53−/−cells. (C) HCT116 cells were plated and treated with U0126 or LY294002 as indicated, 24 h after plating. Cells were irradiated (5 Gy). Cells were re-plated as single cells for colony formation assays, 96 h after irradiation. Colonies were permitted to form over the following 10 days. The numbers of colonies were expressed asa fraction of the respective mock-irradiated cells and plotted in a semi-log graph. Data are the means of six separate dishes (s.e.m., n=3) (*P<0.05 less than corresponding vehicle treated cell value; P<0.05 less than corresponding LY294002 treated cell value).

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