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. Author manuscript; available in PMC: 2012 Jun 28.
Published in final edited form as: Clin Cancer Res. 2008 Feb 15;14(4):1266–1273. doi: 10.1158/1078-0432.CCR-07-1606

Gefitinib Radiosensitizes Non–Small Cell Lung Cancer Cells by Suppressing Cellular DNA Repair Capacity

Toshimitsu Tanaka 1, Anupama Munshi 1, Colin Brooks 1, Jenny Liu 1, Marvette L Hobbs 1, Raymond E Meyn 1
PMCID: PMC3385646  NIHMSID: NIHMS383668  PMID: 18281562

Abstract

Purpose

Overexpression of the epidermal growth factor receptor (EGFR) promotes unregulated growth, inhibits apoptosis, and likely contributes to clinical radiation resistance of non – small cell lung cancer (NSCLC). Molecular blockade of EGFR signaling is an attractive therapeutic strategy for enhancing the cytotoxic effects of radiotherapy that is currently under investigation in preclinical and clinical studies. In the present study, we have investigated the mechanism by which gefitinib, a selective EGFR tyrosine kinase inhibitor, restores the radiosensitivity of NSCLC cells.

Experimental Design

Two NSCLC cell lines, A549 and H1299, were treated with 1 μmol/L gefitinib for 24 h before irradiation and then tested for clonogenic survival and capacity for repairing DNA double strand breaks (DSB). Four different repair assays were used: host cell reactivation, detection of γ-H2AX and pNBS1 repair foci using immunofluorescence microscopy, the neutral comet assay, and pulsed-field gel electrophoresis.

Results

In clonogenic survival experiments, gefitinib had significant radiosensitizing effects on both cell lines. Results from all four DNA damage repair analyses in cultured A549 and H1299 cells showed that gefitinib had a strong inhibitory effect on the repair of DSBs after ionizing radiation. The presence of DSBs was especially prolonged during the first 2 h of repair compared with controls. Immunoblot analysis of selected repair proteins indicated that pNBS1 activation was prolonged by gefitinib correlating with its effect on pNBS1-labeled repair foci.

Conclusions

Overall, we conclude that gefitinib enhances the radioresponse of NSCLC cells by suppressing cellular DNA repair capacity, thereby prolonging the presence of radiation-induced DSBs.

Lung cancer is the leading cause of cancer death worldwide, and non–small cell lung cancer (NSCLC) accounts for 80% of all lung cancers. In spite of treatment advances over the past decade, 2-year survival from this disease is only 25% and 5-year survival is only 17% (1). Obviously, novel therapeutic strategies to improve survival of patients with NSCLC are urgently needed. This need has fueled the increasing interest in therapies that target specific pathways involved in the growth and progression of NSCLC tumors. The epidermal growth factor receptor (EGFR) has been identified as a very attractive target in this regard.

EGFR belongs to the ErbB family of plasma membrane receptor tyrosine kinases and controls many important cellular functions. Activation of EGFR in tumor cells promotes tumor cell proliferation, angiogenesis, invasion, and metastasis and inhibits apoptosis (2). EGFR is overexpressed in many solid tumors, including NSCLC, where ~80% of lung squamous cell carcinomas and approximately half of all lung adenocarcinomas and large-cell carcinomas express high levels of EGFR (3). Earlier studies have indicated that high expression of EGFR correlates with advanced tumor stage, metastasis, and poor prognosis. Previous reports have also suggested that high expression of EGFR is associated with resistance to cancer therapy, including radiotherapy (4). Thus, it seems that alterations in EGFR expression or function may influence the cellular response to ionizing radiation, suggesting that agents that inhibit EGFR signaling would enhance the effectiveness of radiation therapy.

One such agent, gefitinib (ZD1839, “Iressa”; AstraZeneca Pharmaceuticals) is an orally given, small-molecule tyrosine kinase inhibitor that targets the EGFR (5). Gefitinib has proved antitumor efficacy as monotherapy for a subset of patients with NSCLC. Such patients have been shown to have specific mutations in the EGFR gene that confer sensitivity to gefitinib (6). At the same time, ionizing radiation has been shown to induce autophosphorylation of EGFR and activate its downstream signaling pathways, making the two good candidates for combination therapy (7). In fact, several preclinical studies have indicated that gefitinib enhances radiation effects in cell lines or xenograft tumors derived from human colon, ovarian, breast, or NSCLC tumors (8–11). Although the investigations in animal models have suggested that the antitumor activity of gefitinib, in combination with radiation, is partly due to an inhibition of tumor angiogenesis (8, 9), how the drug directly radiosensitizes tumor cells exposed in vitro remains unclear. Understanding the mechanism underlying this effect may help guide the further development of strategies that combine these therapeutics or reveal new molecular targets downstream of EGFR for effectively radiosensitizing NSCLC cells. In the present study, we have tested whether gefitinib radiosensitizes by altering a pathway known to be critical for regulation of cellular response to radiation, i.e., the cells’ ability to efficiently repair radiation-induced lesions in DNA. Four different approaches were taken to assess DNA repair activity: host cell reactivation (HCR), quantification of repair foci containing phosphorylated histone 2AX (γ-H2AX) or phosphorylated Nijmegan breakage syndrome 1 (pNBS1), the neutral comet assay, and pulsed-field gel electrophoresis (PFGE). Use of these assays allowed us to test the ability of gefitinib to affect total DNA repair capacity and repair of radiation-induced double-strand breaks (DSB), specifically.

Materials and Methods

Cell culture

The human NSCLC cell lines A549 and H1299 were obtained from American Type Culture Collection and routinely maintained in RPMI 1640 supplemented with 10% fetal bovine serum, 10,000 units/mL penicillin-streptomycin, and 2 mmol/L L-glutamine. Both of these cell lines have wild-type EGFR (12).

Chemicals

Gefitinib was purchased from the hospital pharmacy. A 10 mmol/L stock solution was prepared in DMSO and stored in aliquots at −20°C until use.

Clonogenic survival

The effectiveness of the combination of gefitinib and ionizing radiation was assessed by clonogenic assays. The NSCLC cell lines were treated with gefitinib at the indicated concentration and then exposed to different doses of ionizing radiation. Briefly, cells were irradiated with a high dose-rate 137Cs unit (4.5 Gy/min) at room temperature in T-25 flasks. After treatment, cells were trypsinized and counted. Known numbers were then replated and returned to the incubator to allow macroscopic colony development. Colonies were counted after ~14 days, and the plating efficiency and surviving fraction for given treatments were calculated based on the survival of nonirradiated cells treated with the vehicle or gefitinib alone.

Immunoblot analysis

Cells were treated with 1 or 2 μmol/L gefitinib for 24 h at 37°C, harvested, rinsed in ice-cold PBS, and lysed in buffer containing 50 mmol/L HEPES (pH 7.9), 0.4 mol/L NaCl, 1 mmol/L EDTA, 2 μg/mL leupeptin, 2 μg/mL aprotinin, 5 μg/mL benzamidine, 0.5 mmol/L phenylmethylsulfonyl fluoride, and 1% NP40. Protein concentration in the lysates was determined by the Bio-Rad Dc protein assay. Equal amounts of protein were separated by 12% SDS-PAGE, transferred to Immobilon (Millipore), and blocked with 5% nonfat dry milk in TBS–Tween 20 (0.05%, v/v) for 1 h at room temperature. The membrane was incubated with primary antibody overnight at 4°C. Antibodies for pNBS1 were obtained from Novus Biologicals; EGFR, pEGFR, AKT, pAKT, extracellular signal-regulated kinase 1/2 (ERK1/2), and pERK1/2 from Cell Signaling; DNA-PK from BD PharMingen; pDNA-PK from Abcam; ataxia telangiectasia mutated (ATM) from Novis Biochemicals; pATM from Calbiochem; and actin from Chemicon. After washing, the membrane was incubated with the appropriate horseradish peroxidase – conjugated secondary antibody (diluted 1:2,000; Amersham Biosciences) for 1 h. The blots were developed by enhanced chemiluminescence (Amersham Biosciences) and visualized on Typhoon 9400 scanner (Amersham Biosciences/GE Healthcare).

HCR

The method used to assay HCR has been described in detail elsewhere (13). Briefly, cells were plated in six-well plates (5 × 104 per well in 3 mL of medium). Twenty-four hours after plating, cells were treated with 1 μmol/L gefitinib for 24 h. Adenovirus carrying the β-galactosidase gene (Ad-βgal, Introgen Therapeutics, Inc.) was irradiated in a centrifuge tube with a high-dose 137Cs unit (4,000 Gy) at room temperature. Control cells and cells treated with gefitinib were infected with irradiated Ad-βgal (A549, 1,000 vector particles per cell; H1299, 100 vector particles per cell) in 1 mL of serum-free medium and incubated for 1 h. Then, 2 mL of complete medium were added to the cells. Twenty-four hours after vector transduction, cells were fixed with 2% formaldehyde and 0.05% glutaraldehyde in PBS and then stained with X-gal overnight. Blue-stained cells (at least 500 per well) were counted, and the percentage of cells that stained was calculated.

Immunofluorescent staining for γ-H2AX and pNBS1

Cells were grown and treated with 1 μmol/L gefitinib for 24 h on coverslips placed in 35-mm dishes. At specified times, medium was aspirated, and cells were fixed in 1% paraformaldehyde for 10 min at room temperature. Paraformaldehyde was aspirated, and the cells were fixed in 70% ethanol for 10 min at room temperature followed by treatment with 0.1% NP40 in PBS for 20 min. Cells were washed in PBS twice and then blocked with 5% bovine serum albumin in PBS for 30 min. Anti–γ-H2AX (Trevigen) or anti-pNBS1 antibody was added at a dilution of 1:300 in 5% bovine serum albumin in PBS, and incubation continued for 2 h at room temperature with gentle shaking. Cells were then washed four times in PBS before being incubated in the dark with a FITC-labeled secondary antibody at a dilution of 1:300 (γ-H2AX) and 1:150 (pNBS1) in 5% bovine serum albumin in PBS for 30 min. The secondary antibody solution was then aspirated, and the cells were washed four times in PBS. Cells then were incubated in the dark with 44′,6-diamidino-2-phenylindole (1 μg/mL) in PBS for 5 min, and coverslips were mounted with an antifade solution (Molecular Probes). Slides were examined on a Leica fluorescence microscope, and images were captured by a CCD camera and imported into Advanced Spot Image analysis software package. For each treatment condition, the number of γ-H2AX or pNBS1 foci were determined in at least 50 cells. All observations were validated by at least three independent experiments.

Neutral comet assay

For detection of radiation-induced DSBs, a CometAssay kit (Trevigen) was used according to the manufacturer’s instructions. Briefly, cells pretreated with 1 μmol/L gefitinib for 24 h were irradiated with 20 Gy after which they were harvested, washed twice in ice-cold PBS, and resuspended at 1 × 105/mL in ice-cold PBS. The cells were then combined with low–melting point agarose at a ratio of 1:10 (v/v) and spread on glass slides (Trevigen). The slides were allowed to solidify for 30 min in the dark at 4°C and were then submerged in precooled, neutral lysis buffer at 4°C for 45 min. After lysis, slides were washed twice in 1× Tris-borate EDTA solution [0.89 mol/L Tris, 0.88 mol/L boric acid, 2 mmol/L EDTA (pH 8.3)] for 20 min each and subjected to electrophoresis at 1.0 V/cm for 20 min. The slides were then rinsed with distilled water and placed in 70% ethanol for 5 min, after which they were left to air dry. The nuclei were then stained with SYBR Green and comet images were obtained with a Leica fluorescence microscope with an attached CCD camera. Images were saved as Bitmap files and analyzed using CometScore software (TriTek). The Olive Tail Moment (14) was determined for 50 cells in each sample.

PFGE

PFGE was done as described previously (15, 16). Briefly, cells pretreated with 1 μmol/L gefitinib for 24 h were irradiated on ice with 40 Gy. Immediately after irradiation, the medium was replaced with warm medium and the cells were placed in a 37°C incubator for the appropriate time for repair. Cells were then trypsinized on ice, washed, and embedded in agarose plugs. The plugs were lysed and digested with proteinase K. DNA fragments were separated using a CHEF-DR III system (Bio-Rad Laboratories) at 1.5 V/cm for 20 h at 25°C in 0.5× Tris-borate EDTA buffer. After electrophoresis, the gel was transferred to a nylon membrane for 3 days at room temperature. The membrane was then hybridized with a 32P-labeled human Alu+ probe for 18 h at 45°C. The fraction of DNA released into the lane and that remaining in the plug was determined on the membrane using a Typhoon 9400 storage phosphor imaging system and ImageQuant software (Amersham Biosciences/GE Healthcare).

Statistical analysis

Data were analyzed using the paired t test and described as mean ± SE. A difference was regarded as significant if P < 0.05.

Results

Treatment with gefitinib enhances radiosensitivity in human NSCLC cells in in vitro clonogenic survival assays

We measured the survival of human NSCLC cells exposed to combinations of gefitinib and ionizing radiation using clonogenic assays. A549 and H1299 cells were pretreated with 1 or 2 μmol/L gefitinib for 24 h, and then the cells were irradiated and plated for clonogenic cell survival. These drug concentrations were chosen to bracket the range of gefitinib levels achieved in the plasma of treated patients (17, 18). Pretreatment with gefitinib suppressed the clonogenic survival of both cell lines (Fig. 1). In A549 cells, survival at 2 Gy (SF2) was reduced from 50.4 ± 0.7% in the control to 39.4 ± 0.8% (P = 0.05) in 1 μmol/L gefitinib–treated cells (Fig. 1A). Similar results were obtained using the H1299 cell line: SF2 was reduced from 55.8 ± 2.5% in the control cells to 40.9 ± 0.06% (P = 0.01) in 1 μmol/L gefitinib–treated cells (Fig. 1B). The radiosensitizing effect of gefitinib was not significantly increased by using 2 μmol/L drug. Dose enhancement factors were calculated by dividing the radiation dose that produced 10% cell survival on the radiation-only survival curve by that for the corresponding gefitinib plus ionizing radiation curve. The dose enhancement factor for the A549 cells was 1.1 and that for H1299 was 1.2 in 1 μmol/L gefitinib–treated cells. Gefitinib, when used alone at either concentration, did not significantly reduce the plating efficiency of A549 or H1299 cells compared with untreated controls.

Fig. 1.

Fig. 1

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Gefitinib radiosensitizes NSCLC cells and suppresses their signaling pathways downstream of EGFR. A549 (A and C) and H1299 (B and D) cells were treated with gefitinib (1 or 2 μmol/L for 24 h) and assessed for radiosensitization by clonogenic cell survival (A and B) immediately after irradiation or harvested for immunoblot analysis (C and D) 2 h after irradiation. For the survival curves, each data point represents the average of three independent experiments each plated in triplicate: solid line, control; dotted line, 1 μmol/L; dashed line, 2 μmol/L. Bar, SE. The effects of gefitinib treatment on the downstream signaling of the EGFR pathway proteins EGFR, pEGFR, AKT, pAKT, ERK, and pERK were analyzed by immunoblot analysis. Actin was used as a loading control.

Effects of gefitinib on downstream signaling of EGFR

To test whether gefitinib targets the EGFR in the cell lines under investigation, we examined the effect of gefitinib treatment on the expression of downstream signals in the EGFR pathway. Decreases in the levels of pEGFR were observed in unirradiated A549 and H1299 cells after a 24-h treatment with 1 or 2 μmol/L gefitinib (Fig. 1C and D), indicating that gefitinib was acting as expected. The levels of pAKT were also lowered in both cell lines when unirradiated, but only after the higher concentration of 2 μmol/L. Radiation activated pEGFR and pAKT in A549 cells but not in H1299 cells, and this activation was suppressed in a concentration-dependent manner by gefitinib in the A549 line. On the other hand, radiation activated pERK in both cell lines, and this activation seemed to be equally suppressed by both concentrations of gefitinib. Interestingly, gefitinib suppressed pERK in the irradiated cells to levels lower than in the unirradiated cells, suggesting that pERK activation becomes considerably more dependent on EGFR signaling after irradiation compared with the basal setting. Considering that there was no significant difference between the effect of 1 and 2 μmol/L gefitinib on loss of clonogenic survival (Fig. 1A and B), the radiosensitizing effect of gefitinib correlates with its ability to suppress pERK activation.

Gefitinib suppresses DNA repair detected using a HCR assay

We did an HCR assay to determine if the radiosensitizing effect of gefitinib seen in Fig. 1 could be explained as a suppression of the total capacity of the NSCLC cells for functionally repairing the spectrum of radiation-induced DNA lesions. For this, A549 and H1299 cells, mock treated or pretreated with gefitinib, were subsequently infected with Ad-βgal that had been either unirradiated or irradiated with 4,000 Gy of γ-radiation. The rather large dose of radiation was required based on the very small genome size of the adenovirus vector compared with a mammalian cell. Although this assay is not specific for DSBs, to put this dose into perspective, we calculated that it should induce approximately one DSB per vector particle. The ability of the NSCLC cells to reactivate the irradiated Ad-βgal on the basis of βgal expression was assessed 24 h later. Gefitinib-pretreated (1 μmol/L for 24 h) A549 (Fig. 2A) and H1299 (Fig. 2B) cells had a significantly (P = 0.04 and P = 0.006, respectively) lower capacity to reactivate irradiated Ad-βgal compared with cells that were not pretreated with gefitinib.

Fig. 2.

Fig. 2

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Gefitinib suppresses HCR in A549 (A) and H1299 (B) cells. Cells (2 × 104) were seeded in each well of six-well plates and were either left untreated or were treated with 1 μmol/L gefitinib for 24 h. They were then infected with unirradiated or irradiated (4,000 Gy) Ad-βgal for another 24 h. The cells were then stained for β-gal, and the β-gal – positive cells were counted and recorded. The percentage of positive cells were normalized to controls for comparison. Columns, average of at least three independent experiments; bars, SE.

Gefitinib prolongs the expression of radiation-induced γ-H2AX and pNBS1 foci

Although the results of the HCR experiments suggested that gefitinib-pretreated cells displayed a suppressed capacity for repairing the spectrum of radiation-induced DNA lesions, it was important to test whether the repair of DSBs, the major lethal lesion, was specifically inhibited. Thus, γ-H2AX and pNBS1 foci were assessed as indicators of radiation-induced DSBs. γ-H2AX and pNBS1 foci could be clearly distinguished soon after irradiation (2 Gy) of A549 and H1299 cells. The average number of γ-H2AX and pNBS1 foci per cell were assessed beginning at 30 min after 2 Gy and followed thereafter for 24 h (Fig. 3). The average number of γ-H2AX foci per cell in cultures receiving the combined gefitinib/radiation treatment was significantly greater than in the radiation-only group at the 30-min time points for the A549 and H1299 cell lines, P = 0.035 and P = 0.003, respectively (Fig. 3A and C). The average number of pNBS1 foci per cell followed a pattern similar to that seen for to γ-H2AX foci, except that the average number of foci per cell peaked at 1 h (Fig. 3B and D). In addition, the average number of pNBS1 foci per cell in cells receiving the combined gefitinib/radiation treatment was significantly greater than in the radiation-only group at 30 min (P = 0.01) in A549 cells and at 1 h (P = 0.01) in the H1299 cells (Fig. 3B and D). Treatment with gefitinib alone produced no significant induction of γ-H2AX or pNBS1 foci. This prolongation of γ-H2AX and pNBS1 foci levels after the combination compared with controls suggests that gefitinib-mediated radiosensitization may involve a suppression of DSB repair pathways primarily during the early phase of DSB repair.

Fig. 3.

Fig. 3

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Gefitinib suppresses the repair of radiation-induced DSBs detected on the basis of γ-H2AX and pNBS1 foci. A549 (A and B) and H1299 (C and D) cells growing on coverslips in 35-mm dishes were exposed to gefitinib (1 μmol/L) for 24 h, irradiated (2 Gy), and fixed at the specified times for analysis of nuclear γ-H2AX and pNBS1 foci using immunofluorescence microscopy. Columns, average of three independent experiments; bars, SE.

Gefitinib delays the rejoining of radiation-induced DSBs detected by the neutral comet assay or PFGE

An alternative interpretation of the findings presented in Fig. 3 would be that instead of prolonging the presence of DSBs at early times after irradiation, gefitinib instead causes an enhanced number of initial radiation-induced breaks at time 0. The repair foci assay is not amenable to assessing the initial number of DSBs because it takes a finite amount of time for foci to form. Thus, we did an independent assessment of DSB induction and repair after gefitinib treatment using two different assays, the neutral comet assay and PFGE. As shown in Fig. 4, for both cell lines using both assays, a pretreatment with gefitinib did not, within experimental error, alter the initial level of DSBs induced when measured immediately after irradiation. However, again for both cell lines and both assays, this analysis confirmed that, in gefitinib pretreated cells, DSB rejoining is delayed during the first 2 h after irradiation. In every case, the level of DSBs were higher than controls in the gefitinib-pretreated cells after 1 and 2 h of repair, and these differences were statistically significant at the P < 0.05 level. A low level of residual unrejoined DSBs could be detected 24 h after irradiation in some instances, and although these seemed to be enhanced by gefitinib, these differences did not reach statistical significance.

Fig. 4.

Fig. 4

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Gefitinib suppresses the repair of radiation-induced DSBs detected on the basis of the comet assay and PFGE. A549 (A and C) and H1299 cells (B and D) were exposed to gefitinib (1 μmol/L) for 24 h, irradiated with either 20 Gy for the comet assay (A and B) or 40 Gy for PFGE (C and D) and harvested at the indicated times. Columns, average of three independent experiments; bars, SE.

Gefitinib alters the kinetics of pNBS1 activation

Additional experiments were carried out in an attempt to uncover what aspect of molecular repair is altered in gefitinib-treated cells. Previous reports in the literature (19, 20) have indicated that targeting the EGFR with the monoclonal antibody C225 inhibits translocation of EGFR to the nucleus where it would otherwise participate in the activation of the DNA repair protein DNA-PK after irradiation. In addition, other investigators have reported that gefitinib alters the subcellular distribution of DNA-PK leading to suppressed levels of DNA-PK in the nucleus after irradiation (21–23). To test whether these effects of EGFR targeting were operative in the context of our studies, we examined the nuclear levels of EGFR and DNA-PK after gefitinib treatment in A549 and H1299 cells. Immunoblot analysis showed that gefitinib pretreatment did not alter the nuclear levels of either EGFR or DNA-PK 30 min after irradiation in either cell line (Fig. 5A). Moreover, because of the importance of radiation-induced activation of DNA-PK and ATM in the initiation of DSB repair, activation of these repair proteins was also assessed. Gefitinib pretreatment did not alter the radiation-induced activation of either DNA-PK or ATM at this time point. Because our analysis of pNBS1 foci shown in Fig. 3 indicated that the kinetics of this activation of protein may be altered in gefitinib-treated cells, we assessed the activation of pNBS1 using immunoblot analysis. As shown in Fig. 5B, for both cell lines, gefitinib seemed to enhance the robustness of and prolong the pNBS1 signal after irradiation, confirming what was seen at the level of pNBS1 foci.

Fig. 5.

Fig. 5

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Gefitinib does not affect the nuclear levels of EGFR, DNA-PK, ATM, or their activated forms after irradiation but does enhance the activation of NBS1. A549 and H1299 cells were exposed to gefitinib (1 μmol/L) for 24 h, irradiated or not with 5 Gy, and harvested 30 min after irradiation for the results in A or at the indicated times for B. Actin served as a loading control.

Discussion

Radiation therapy remains an important part of the treatment regimen for NSCLC, especially for patients with unresectable tumors where concurrent chemotherapy is usually added. Agents, such as gefitinib, that target the specific molecular defects associated with NSCLC may improve the outcome of these patients when added to conventional modalities. Here, we have shown that gefitinib, at the clinically achievable level of 1 μmol/L for 24 h, sensitizes NSCLC cell lines to radiation, confirming prior reports where this combination was tested on cell lines and xenograft tumors derived from a variety of human cancers, including NSCLC (8–11). Although the degree of in vitro radiosensitization might seem to be small, the ~25% increase in cell kill after the dose of 2 Gy would be expected to have a substantial clinical effect if repeated over the entire course of radiotherapy, i.e., 30 or more fractions of 2 Gy, with daily administration of gefitinib. This enhancement of the intrinsic tumor cell radiosensitivity would be in addition to the previously described effects of gefitinib on tumor angiogenesis (8, 9) that would also contribute to tumor radiation response in vivo.

However, in spite of a growing interest in combining EGFR-targeting agents and radiation as a clinical strategy for treating NSCLC and other cancers and the preclinical findings referenced above that support this concept, the molecular mechanism by which agents, such as gefitinib, mediate their radiosensitizing effects is not known. Generally, radiosensitivity is governed by the capacity of the cell for efficient repair of radiation-induced lesions in the DNA, mainly the repair of DSBs. There have been indications in the literature that some agents that target EGFR may radiosensitize by partly suppressing key components of the DNA repair pathways (19–26).

Thus, based on these reports, we tested whether gefitinib could affect the actual processing of radiation-induced DNA lesions using four different approaches. We used HCR, a relatively old technique that has been modernized by delivering a reporter gene using an adenovirus (27). Irradiation of this vector is expected to reproduce the entire spectrum of DNA lesions that would be seen in the cellular genome, but we specifically irradiated the vector with a dose that would induce one DSB per vector particle on average. We used this technique in two prior studies, where we validated its ability to detect cellular deficiencies in repair (13, 16). Here, we show that a pretreatment of both the A549 and H1299 cells with gefitinib suppresses the ability of those cells to reactivate the irradiated vector compared with control cells that were not treated with gefitinib. This result was consistent with a suppression of cellular DNA repair capacity by gefitinib but does not focus directly on repair of DSBs, the radiation-induced lesions most critical for cell killing. Therefore, in the second approach, we directly tested for an effect of gefitinib on repair of DSBs by measuring the formation and persistence of repair foci. These foci were detected after irradiation using immunofluorescent staining for γ-H2AX and pNBS1, two proteins that aggregate at the sites of DSBs and, along with several other repair proteins, facilitate their repair (28). Our results show, for both A549 and H1299 cells, that the number of radiation-induced γ-H2AX–stained and pNBS1-stained foci are higher in gefitinib-treated cells compared with controls. This is evident at times up to 1 h after irradiation, the time frame where the bulk of DSBs are repaired. Thus, this persistence of foci in the gefitinib-treated cells is interpreted as an inhibition of the DSB repair pathway. Recent reports indicate that persistence of repair foci correlates with enhanced radiosensitivity (29, 30). To confirm that these foci measurements depict inhibition of DSB repair, we did two additional assays, the neutral comet assay and PFGE. Both of these assays that are also specific for DSBs confirmed the observation that repair of radiation-induced DSBs is suppressed in gefitinib-pretreated cells.

The remaining question is how gefitinib produces this effect. By suppressing EGFR signaling, gefitinib inhibits two important downstream pathways, the phosphoinositide 3-kinase/AKT and Ras-Raf-Mek-ERK signaling cascades (2). Both of these pathways have been shown to induce radio-resistance when activated, and their suppression is expected to radiosensitize. Here, we show that in the A549 and H1299 cells, gefitinib suppressed radiation-induced pAKT in the A549 cells but not in the H1299 cells but suppressed radiation-induced activation of pERK in both cell lines. Thus, gefitinib may mediate its effects on DNA repair pathways via a suppression of ERK activation. The ERK pathway has long been considered a major factor in producing radioresistance possibly through its ability to up-regulate the transcription of DNA repair genes when activated (31). Moreover, two recent papers illustrate direct relationships between the ERK pathway and the ATM kinase (32, 33). In our experiments, however, gefitinib did not seem to affect the radiation-induced activation of ATM (Fig. 5A).

An alternative explanation relates to the previous indications that some agents that target EGFR suppress the cellular distribution or expression of certain proteins involved in DNA repair (19–26). Notable in this regard are the reports that C225, a humanized antibody that binds to the EGFR and blocks its signaling, causes a redistribution of DNA-PK from the nucleus to the cytosol and blocks the transport of EGFR to the nucleus (19, 20, 24). Our results suggest that gefitinib may work differently from C225, as we did not observe changes in the nuclear levels of either EGFR or DNA-PK after gefitinib treatment. In another study, gefitinib was reported to lower the nuclear levels of DNA-PK after irradiation (22). However, these effects were seen using concentrations of 10 μmol/L gefitinib, a dose much higher than that used here. Shintani et al. (21) reported that a dose of 1 μmol/L gefitinib suppressed nuclear DNA-PK levels in head and neck squamous cell carcinoma cells, but we did not observe this in the NSCLC cells tested here. The only change in a DNA repair protein that we were able to observe involved pNBS1, where we saw a more robust and prolonged activation after irradiation in gefitinib-pretreated cells compared with controls. Whereas one might assume that a more robust activation of pNBS1 would lead to a more robust repair, this may not necessarily be the case. Indeed, Rhee et al. (34) have recently shown that even a modest overexpression of NBS1 produces a radiosensitizing effect. They speculate that because NBS1 functions in a protein complex, an abnormal abundance of NBS1 may sequester other repair proteins away from the sites of DSBs. At present there is no obvious connection between any of the signaling pathways downstream of EGFR and pNBS1. However, because NBS1 is a substrate for the ATM kinase, a possible connection between EGFR>ER-K>ATM>NBS1 should be examined in further studies.

In summary, we have shown that the EGFR tyrosine kinase inhibitor gefitinib, at pharmacologically achievable levels, prolongs the presence of unrepaired DSBs after irradiation correlating with its radiosensitizing property. Whereas the mechanism of this repair inhibition is not revealed in this investigation, this finding suggests an intersection between one or more of the signaling pathways downstream of the receptor and the regulation of DSB repair in the nucleus. Further examination of these interactions may reveal additional strategies for radiosensitizing human tumor cells or uncover biomarkers for identifying patients who may benefit from the combination of molecularly targeted agents and radiotherapy.

Acknowledgments

Grant support: Department of Defense grant W81XWH-05-2-0027 (R.E. Meyn), National Cancer Institute grants PO1CA06294 (R.E. Meyn), P50 CA070907 (A. Munshi), and P30 CA016672, Kathryn O’Connor Research Professorship (R.E. Meyn).

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