REPORT

Epidermal Growth Factor Receptor as a Genetic Therapy Target for Carcinoma Cell Radiosensitization

Guido Lammering, Theodore H. Hewit, William T. Hawkins, Joseph N. Contessa, Dean B. Reardon, Peck-Sun Lin, Kristoffer Valerie, Paul Dent, Ross B. Mikkelsen, Rupert K. Schmidt-Ullrich

Affiliations of authors: G. Lammering, Department of Radiation Oncology, Medical College of Virginia Campus, Virginia Commonwealth University, Richmond, and Department of Radiation Oncology, Heinrich-Heine University, Duesseldorf, Germany; T. H. Hewit, W. T. Hawkins, J. N. Contessa, P.-S. Lin, K. Valerie, P. Dent, R. B. Mikkelsen, R. K. Schmidt-Ullrich, Department of Radiation Oncology, Medical College of Virginia Campus, Virginia Commonwealth University; D. B. Reardon, School of Pharmacy, University of Louisiana, Monroe.

Correspondence to: Rupert K. Schmidt-Ullrich, M.D., Department of Radiation Oncology, Medical College of Virginia Campus, Virginia Commonwealth University, P.O. Box 980058, 401 College St., Richmond, VA 23298–0058 (e-mail: rullrich{at}hsc.vcu.edu).


    ABSTRACT
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 Notes
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: Exposure of human cancer cells to ionizing radiation activates the epidermal growth factor receptor (EGFR), which, in turn, mediates a cytoprotective response that reduces the cells' sensitivity to ionizing radiation. Overexpression of a dominant-negative EGFR mutant, EGFR-CD533, disrupts the cytoprotective response by preventing radiation-induced activation of the receptor and its downstream effectors. To investigate whether gene therapy with EGFR-CD533 has the potential to increase tumor cell radiosensitivity, we introduced an adenoviral vector containing EGFR-CD533 into xenograft tumors in nude mice and evaluated the tumor response to ionizing radiation. Methods: Xenograft tumors established from the human mammary carcinoma cell line MDA-MB-231 were transduced via infusion with the adenoviral vector Ad-EGFR-CD533 or a control vector containing the {beta}-galactosidase gene, Ad-LacZ. The transduced tumors were then exposed to radiation in the therapeutic dose range, and radiation-induced EGFR activation was assessed by examining the tyrosine phosphorylation of immunoprecipitated EGFR. Radiosensitization was determined in vitro by colony-formation assays. All statistical tests were two-sided. Results: The transduction efficiency of MDA-MB-231 tumors by Ad-LacZ was 44%. Expression of EGFR-CD533 in tumors reduced radiation-induced EGFR activation by 2.94-fold (95% confidence interval [CI] = 2.23 to 4.14). The radiosensitivity of Ad-EGFR-CD533-transduced tumors was statistically significantly higher (46%; P<.001) than that of Ad-LacZ-transduced tumors, yielding a dose-enhancement ratio of 1.85 (95% CI = 1.54 to 2.51). Conclusions: Transduction of MDA-MB-231 xenograft tumors with Ad-EGFR-CD533 conferred a dominant-negative EGFR phenotype and induced tumor radiosensitization. Therefore, disruption of EGFR function through overexpression of EGFR-CD533 may hold promise as a gene therapeutic approach to enhance the sensitivity of tumor cells to ionizing radiation.



    INTRODUCTION
 Top
 Notes
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The epidermal growth factor receptor (EGFR) and other ErbB receptor tyrosine kinases (RTKs) are involved in the autocrine growth regulation of many carcinoma cells (14). We have demonstrated that the exposure of tumor cells to ionizing radiation in the therapeutic dose range (i.e., 1–5 Gy) results in the immediate activation of EGFR (57) and that repeated radiation exposures of 2 Gy lead to increased EGFR expression (8). The activation of EGFR by radiation (5,9) was defined by a several-fold increase in tyrosine phosphorylation currently indistinguishable from the effects induced by the epidermal growth factor (5,912). In addition, radiation-induced EGFR activation results in a pronounced radiation dose-dependent, proliferative response that can be quantified in vitro after both single (10) and repeated (6,13) radiation exposures. On the basis of these findings, we have concluded that radiation-induced EGFR activation contributes, at least in part, to the mechanism of accelerated proliferation (11,1416). This cellular proliferation response during repeated radiation exposures, as used in clinical radiotherapy (1618), leads to increased renewal of tumor clonogens (16,19,20). In addition, the increased biosynthetic activity of rapidly proliferating tumor cells can be expected to increase their capacity for DNA damage repair. Because both the proliferative and the DNA repair responses counteract the toxic effects of radiation therapy, we have defined them as cytoprotective. Considering the role of EGFR in initiating these responses, disruption of EGFR function should prevent the cytoprotective responses and mediate tumor cell radiosensitization.

To examine the broad therapeutic potential of inhibiting EGFR activation after irradiation, we have studied the conditions for optimal radiosensitization in a spectrum of human tumor cells that are autocrine growth regulated by EGFR (6,7,13). For reasons of specificity and broad applicability, we chose to inhibit EGFR through a genetic approach by overexpressing EGFR-CD533, a mutant of EGFR that lacks the entire cytoplasmic domain of 533 amino acids and confers no transformation or proliferation-promoting activity (21,22). EGFR-CD533 disrupts the function of the entire ErbB RTK network through receptor–protein interactions independent of the varied receptor-expression profiles seen in different tumor cells (22,23). Induction of EGFR-CD533 expression in stably transfected MDA-EGFR-CD533 mammary carcinoma cells completely blocked cytoprotective, proproliferative signaling along the EGFR/mitogen-activated protein kinase (MAPK) cascade, including the radiation-induced activation of EGFR and MAPK, and blocked the stimulation of cell proliferation (6,13). These inhibitory effects are associated with marked radiosensitization of these tumor cells in vitro [(6,7); Lammering G, Lin P-S, Schmidt-Ullrich RK: unpublished data]. Because two underlying mechanisms can be expected to contribute to in vivo radiosensitivity examined in this study, we propose inhibition of radiation-induced proliferation and direct radiosensitization (6,7).

The current studies examine, in preclinical experiments, whether expression of EGFR-CD533 can confer radiosensitization in vivo. The tumor models use xenograft tumors, grown in nude mice, of the human mammary carcinoma cell MDA-MB-231 and the stably transfected counterpart MDA-EGFR-CD533 (6). We use a genetic therapy approach of tumor cell transduction in vivo, whereby a replication-incompetent adenoviral vector expressing EGFR-CD533, Ad-EGFR-CD533, is infused into established MDA-MB-231 tumors. The studies presented include the extent of radiosensitization conferred by EGFR-CD533 expression as quantified by ex vivo (in vivo/in vitro) clonogenic survival analyses that compare MDA-MB-231 cells isolated from tumors transduced in vivo with Ad-EGFR-CD533 with cells from MDA-EGFR-CD533 tumors, in which all of the cells are engineered to stably overexpress EGFR-CD533. The studies are complemented by immunochemical analyses and kinase assays establishing mechanistic links between inhibition of radiation-induced EGFR activation by EGFR-CD533 and tumor cell radiosensitization.


    MATERIALS AND METHODS
 Top
 Notes
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Cell Culture

The human mammary carcinoma cell line MDA-MB-231 was obtained from the American Type Culture Collection (Manassas, VA). The MDA-TR15-EGFR-CD533 cell line, referred to as MDA-EGFR-CD533, was developed in our laboratory by stably transfecting MDA-MB-231 cells with a plasmid containing the EGFR-CD533 complementary DNA (cDNA), the expression of which is under the control of a doxycycline-inducible promoter (6). Both of the cell lines were maintained in RPMI-1640 medium containing 5% tetracycline-free fetal calf serum (RPMI/5FCS) and antibiotics (penicillin/streptomycin), except when the cells were exposed to RPMI-1640 medium containing 0.5% FCS (RPMI/0.5FCS) before irradiation as described previously (6,13).

Animals and Tumors

Athymic female NCr-nu/nu mice were obtained from the Animal Production Area, National Cancer Institute-Frederick Cancer Research Facility (Frederick, MD). Mice were maintained under pathogen-free conditions in facilities approved by the American Association for Accreditation of Laboratory Animal Care and in accordance with current regulations and standards of the U.S. Department of Agriculture, Washington, DC, the U.S. Department of Health and Human Services, Washington, DC, and the National Institutes of Health, Bethesda, MD. Single tumors were produced by subcutaneously injecting 107 viable MDA-MB-231 or MDA-EGFR-CD533 cells into the right hind leg of 4- to 6-week-old mice. Tumors derived from MDA-MB-231 cells reached a tumor size of 8–10 mm after 4–6 weeks, whereas tumors from MDA-EGFR-CD533 reached the desired tumor size after 12–16 weeks. All experiments were conducted with tumors 8–10 mm in diameter.

Generation of Adenoviral Vectors

A replication-incompetent adenovirus was produced as described previously (24,25). Adenoviral vectors containing EGFR-CD533 cDNA, Ad-EGFR-CD533, or the bacterial lacZ reporter gene as a control, Ad-LacZ, were produced in HEK 293 cells and purified as described previously (25,26). The dominant-negative EGFR-CD533 cDNA (12) was provided by A. Ullrich (Max-Planck-Institute for Biochemistry, Martinsried, Germany).

Transduction Protocols and Assessment of Transduction Efficiency

MDA-MB-231 cells were seeded at a density of 1.4 x 105 cells in 60-mm2 dishes and were cultured in RPMI/5FCS for 5 days. On day 3, MDA-MB-231 cells were transduced with Ad-EGFR-CD533 or Ad-LacZ at a multiplicity of infection (MOI) of 50 or were mock transduced with an equivalent volume of media that did not contain any adenovirus. We quantified the transduction efficiency 48 hours after transduction with Ad-LacZ by staining the cells for {beta}-galactosidase, counterstaining with safranin O, and then counting the number of {beta}-galactosidase-positive cells from a total of 500 cells (26,27). The MOI was optimized for maximum transduction and minimum cellular toxicity, so that there was less than a 20% reduction in colony-formation efficiency relative to mock-transduced cells.

MDA-MB-231 tumor xenografts were infused with Ad-LacZ or Ad-EGFR-CD533 vectors by a constant flow rate controlled at approximately 0.5 µL/minute by use of a positive pressure-infusion device (26,28). Initial experiments delivered the adenoviral vectors, 1010 plaque-forming units (pfu) in 0.12 mL of phosphate-buffered saline (PBS), over a 40-minute period. To optimize the adenoviral vector delivery, we compared transduction efficiencies after single 4- or 6-track infusions, by the use of one 30-gauge needle/track, with repeated infusions of the 6-track arrangement on 2 consecutive days (2 x 6 track). The most efficient transduction in vivo was achieved with the 2 x 6-track setup that placed two sets of three needles in opposing directions that penetrated 60% of the tumor diameter. The needles were spaced evenly to achieve the widest possible distribution of adenovirus delivery within the tumor, and each needle was retracted by 1 mm every 10 minutes during the 40-minute infusion. The 2 x 6-track method was repeated as a second 6-track infusion on the second day, with the exception that the needles penetrated the tumor at right angles to the initial 6-track infusion (see Fig. 3Go, B). A Bee Hive Controller and a Baby Bee Syringe Pump (Bioanalytical Systems, Inc., West Lafayette, IN) were used for all adenoviral infusions. All infusions were performed on fully anesthetized mice.



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Fig. 3. Optimization of intratumoral infusion of adenoviral vectors into MDA-MB-231 xenograft tumors and expression of EGFR-CD533 in these tumors. A) MDA-MB-231 tumors were transduced in vivo by different protocols (4, 6, and 2 x 6 track) for intratumoral infusion with the same quantity (1010 plaque-forming units) of an adenoviral vector containing the bacterial LacZ gene (Ad-LacZ). Tumors were isolated 48 hours after infusion, and the transduction efficiency was determined in single-cell suspensions by quantifying the percentage of {beta}-galactosidase ({beta}-gal)-positive tumor cells. Data represent the mean number of {beta}-gal-positive cells and 95% confidence intervals from three independent experiments for each infusion protocol. B) Schematic depiction of the 2 x 6-track treatment protocol for experiments testing the in vivo radiosensitizing effects of intratumoral infusion of the adenoviral vector Ad-EGFR-CD533 by use of the MDA-MB-231 xenograft tumors. The 2 x 6-track infusions of Ad-EGFR-CD533 were performed on consecutive days, and the mice were subsequently irradiated with 1.5 Gy on 3 consecutive days (3 x 1.5 Gy; days 4–6). Tumors were harvested, and single cells were generated for colony-formation assays 24 hours after the last irradiation. C) Induction of EGFR-CD533 expression in MDA-MB-231 tumor cells after intratumoral 2 x 6-track infusions of Ad-EGFR-CD533. EGFR and EGFR-CD533 levels in vivo from frozen tumor tissue were assessed by immunoprecipitation and immunoblotting and compared with the in vitro levels (left panel). EGRF and EGFR-CD533 levels were also assessed 96 hours after transfer of the isolated tumor cells to tissue culture (ex vivo/in vitro; Ad-LacZ versus Ad-EGFR-CD533, right panel). Western blots were probed with an anti-epidermal growth factor receptor (EGFR) monoclonal antibody. This antibody also appears to detect the constitutively active truncated EGFR (EGFRvIII) on the basis of the predicted molecular weight (*).

 
Induction of EGFR-CD533 Expression in the Stably Transfected MDA-EGFR-CD533 Cells

MDA-EGFR-CD533 cells were seeded at a density of 1.4 x 105 cells in 60-mm2 dishes and were cultured in RPMI/5FCS for 5 days. On day 3, EGFR-CD533 expression was induced in MDA-EGFR-CD533 cells with doxycycline at 1 µg/mL for 48 hours. EGFR-CD533 expression was assessed as described below.

EGFR-CD533 expression in MDA-EGFR-CD533 tumor xenografts was induced by doxycycline injections. Two protocols were used, depending on the radiation therapy that followed. In one protocol, seven intraperitoneal injections at 0.3 mg at 1 µg/µL of doxycycline were given approximately 12 hours apart over a 90-hour time period. The mice were then irradiated with a 4-Gy single radiation exposure. In the second protocol, twice-daily doxycycline injections were initiated 24 hours before the first radiation exposure and were continued for the consecutive 3 days during which the tumors were irradiated with 1.5 Gy each day. Doxycyline injections continued until 20 hours after the last radiation exposure, at which time the tumors were harvested for processing to single-cell suspensions (see below).

Irradiation Protocols

MDA-MB-231 and MDA-EGFR-CD533 cells were seeded at specified densities and irradiated 5 days thereafter by use of a 60Co source at a dose rate of 1.9 Gy/minute. For all experiments, cells were maintained at 37 °C except for the irradiation itself, which was performed at 20 °C.

The radiosensitizing effects of EGFR-CD533 in vivo in response to ionizing radiation were tested after intratumoral infusion of Ad-EGFR-CD533 in MDA-MB-231 tumors or in MDA-EGFR-CD533 tumors after doxycycline treatment of mice. Radiation exposures in vivo started 2 days after infusion of the adenoviral vectors or were scheduled between the twice-daily doxycycline injections described above. The mice were placed in a plastic container to ensure that they would be exposed to the full radiation dose and were then subjected to whole-body irradiation at 1.5 Gy/day for 3 consecutive days. Controls included mock irradiation of tumors infused with Ad-LacZ or Ad-EGFR-CD533.

For immunochemical verification of radiation-induced EGFR activation, as assessed by the extent of EGFR tyrosine phosphorylation, mice were exposed to a single whole-body radiation dose of 4 Gy. Tumors were excised 2, 5, and 10 minutes after irradiation or 10 minutes after mock irradiation, were frozen immediately in liquid nitrogen, and were processed as described below.

Immunoprecipitation and Immunoblotting Assays

Protein-expression levels of EGFR and EGFR-CD533 and EGFR tyrosine phosphorylation were quantified by immunoprecipitation and western blot analyses. MDA-MB-231 and MDA-EGFR-CD533 cells were maintained in RPMI/0.5FCS for 16 hours before EGF treatment at 100 ng/mL for 5 minutes or irradiation with 4 Gy. Thereafter, cells were processed as described previously (6,13,14).

EGFR-CD533 analysis in tumor tissue was determined in extracts from tumors instantly frozen in liquid nitrogen after excision. Tumor tissue was pulverized in liquid nitrogen by use of a mortar and pestle (29) and immediately lysed in ice-cold lysis buffer (i.e., 25 mM {beta}-glycerophosphate, 25 mM Tris–HCl [pH 7.4], 10% [vol/vol] glycerol, 1.5 mM EGTA, 0.5 mM EDTA, 1% Triton X-100, 1 mM sodium orthovanadate, 1 mM sodium pyrophosphate, 15 µg/mL of aprotinin, 15 µg/mL of leupeptin, 2 µM benzamidine, and 150 µg/mL of phenylmethylsulfonyl fluoride). The cell suspension was passed sequentially through 16- to 21-gauge needles to facilitate cell lysis. Cell debris was removed by centrifugation at 14 000g at 4 °C for 10 minutes, and the supernatants were processed for immunoprecipitation and western blot analysis for EGFR and EGFR-CD533 protein expression and for EGFR tyrosine phosphorylation as described previously (6,13). Autoradiographs were quantified by use of Sigma Scan software (Jandel Scientific, San Rafael, CA) (5).

MAPK Assay

MAPK activity was quantified in MDA-MB-231 cell lysates by use of the immune complex MAPK assay (13). Briefly, lysate aliquots with equivalent amounts of protein, quantified by a Bio-Rad protein assay kit (Bio-Rad Laboratories, Richmond, CA), were incubated with an anti-ERK2 (C-14) antibody (Santa Cruz Biotechnology, Santa Cruz, CA) at 4 °C for 3 hours and washed twice with lysis buffer and once with kinase buffer (13). The immune complexes were incubated with 20 µg of a substrate protein, myelin basic protein (MBP), in the presence of [{gamma}-32P]adenosine-5`-triphosphate (5000 cpm/pmol) at 37 °C for 30 minutes, and the MBP labeling by MAPK was quantified by use of P81 assay paper and liquid scintillation spectroscopy (13).

Clonogenic Survival Assay

In vitro radiosensitivity assays were performed 48 hours after adenoviral vector transduction of MDA-MB-231 cells or doxycycline treatment of MDA-EGFR-CD533 cells. Cells were irradiated as monolayers with single radiation doses of 1, 2, 3, 4, and 8 Gy, maintained in standard culture conditions for 24 hours, then detached with trypsin and plated for colony-formation assays to assess clonogenic survival (30). The number of cells plated in 60-mm2 dishes was adjusted to yield 50–300 colonies per radiation dose. Cells were cultured for 12 days, fixed in methanol, and stained with 0.5% crystal violet, and colonies containing 50 or more cells were counted to determine the surviving fraction of clonogenic cells. The log survival was adjusted to the number of cells plated, after correction for plating efficiency. Relative radiosensitivities were computed by determining the mean inactivation dose () for each treatment group (31). Survival curves were fitted by use of the linear-quadratic model (32).

Ex Vivo Clonogenic Survival Assay

The in vivo radiosensitivity of xenograft tumors was quantified by an ex vivo clonogenic survival assay (33). Mice were killed 24 hours after irradiation, tumors were excised, and single-cell suspensions were generated by digestion with 1.5 mL of collagenase Type IA (280 U/mL) and 1.5 mL of an enzyme cocktail containing deoxyribonuclease (31 U/mL), pronase (225 U/mL), and collagenase Type IA (3280 U/mL) in a total volume of 15 mL of RPMI/5FCS and antibiotics. After 2–3 hours of being stirred at 37 °C, the suspension was passed through a 40-µm mesh filter. The isolated single cells were washed twice with PBS, counted, and plated in quadruplicate for colony formation. The data shown represent the mean values of the clonogenic survival for each tumor.

Statistical Analysis

All data are shown as means and 95% confidence intervals (CIs), unless otherwise specified. Statistical comparisons between clonogenic survival curves were carried out by use of the F test. Student's t test was applied for all other statistical evaluations. The radiation dose-enhancement ratio (DER) under conditions of EGFR-CD533 expression was derived from the ratio of the D values for the control survival curves, Ad-LacZ for MDA-MB-231 cells or for MDA-EGFR-CD533 cells not treated with doxycycline, and the survival curves for EGFR-CD53 expressing corresponding cells in single-dose clonogenic survival assays. The DER in ex vivo clonogenic survival assays after repeated radiation exposures in vivo was derived from the fold difference in clonogenic survival for the control conditions with Ad-LacZ transduction or without doxycycline treatment, relative to Ad-EGFR-CD533 transduction or doxycycline treatment, respectively. A P<.05 was considered to be statistically significant. All P values reported were two-sided. All statistical analyses were carried out with the SAS software package (version 8.0; SAS Institute, Inc., Cary, NC).


    RESULTS
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 Notes
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Transduction of MDA-MB-231 Cells With Ad-EGFR-CD533 and Radiation-Induced Activation of EGFR and MAPK In Vitro

To determine the susceptibility of MDA-MB-231 cells to transduction with adenovirus, we first established the optimal transduction conditions by use of the Ad-LacZ reporter virus. When MDA-MB-231 cells were transduced at 60%–80% confluence with Ad-LacZ at an MOI of 50, the transduction efficiency was greater than 80%, as determined by staining for {beta}-galactosidase 48 hours after transduction (Fig. 1Go, A). Transduction of the MDA-MB-231 cells at an MOI of 50 yielded less than a 20% reduction in colony-formation ability relative to mock-transduced cells (data not shown). Western blot analysis of cell lysates isolated from cells transduced with Ad-EGFR-CD533 showed detectable levels of EGFR-CD533 expression after 24 hours that continued to increase up to 48 hours (Fig. 1Go, B). EGFR-CD533 protein expression levels remained high, at least up to 72 hours (data not shown).



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Fig. 1. Transduction efficiency of the adenoviral vector Ad-EGFR-CD533 at increasing multiplicity of infection (MOI), kinetics of EGFR-CD533 expression, and effects of EGFR-CD533 on radiation-induced epidermal growth factor receptor (EGFR) and mitogen-activated protein kinase (MAPK) activation in vitro. A) MDA-MB-231 cells were transduced with an adenoviral vector containing the bacterial LacZ gene (Ad-LacZ) at MOIs between 5 and 100. The transduction efficiency was quantified as the percentage of {beta}-galactosidase ({beta}-gal)-positive cells after 48 hours. All subsequent experiments used an MOI of 50 for the transduction of cells with an adenoviral vector. B) MDA-MB-231 cells were transduced with Ad-EGFR-CD533 (MDA-Ad-EGFR-CD533). EGFR-CD533 protein-expression levels were determined by immunoblotting and compared with the level of wild-type EGFR at the indicated time points after transduction (transduction at 0 hours). C) MDA-MB-231 cells were mock transduced (MDA-MB-231) or transduced with Ad-LacZ (MDA-Ad-LacZ) or Ad-EGFR-CD533 and 48 hours later were exposed to a single radiation dose of 2 Gy or epidermal growth factor (EGF) (100 ng/mL). EGFR tyrosine phosphorylation (EGFR Tyr-P) was quantified by immunoblotting at the indicated time points after irradiation or after EGF treatment. The corresponding values of EGFR Tyr-P represent fold changes relative to the respective unirradiated control (0 minutes). The results shown are representative of three independent experiments. CI = confidence interval. D) MDA-MB-231 cells were transduced with Ad-LacZ or Ad-EGFR-CD533 and 48 hours later were exposed to a radiation dose of 2 Gy. MAPK activity was quantified from cell lysates at the indicated times after radiation exposure. The corresponding fold increases of MAPK activity represent changes over the unirradiated respective control (0 minutes). The result shown is representative of two independent experiments.

 
We next assessed whether a single radiation exposure could activate the endogenous EGFR in MDA-MB-231 cells transduced with the adenoviral vectors. Mock-transduced MDA-MB-231 cells had a 2.5-fold (95% CI = 1.7 to 3.2) increase in EGFR activation within 2 minutes of a 2-Gy radiation exposure, as determined by an increase in EGFR tyrosine phosphorylation (Fig. 1Go, C). MDA-MB-231 cells transduced with Ad-LacZ had a similar radiation-induced EGFR activation profile (Fig. 1Go, C). On the other hand, in MDA-MB-231 cells transduced with Ad-EGFR-CD533, the radiation-induced EGFR activation was completely inhibited (Fig. 1Go, C). We also assessed EGFR activation in response to EGF in the transduced cells. Compared with MDA-MB-231 cells transduced with Ad-LacZ, EGF-induced EGFR activation was reduced substantially in MDA-MB-231 cells transduced with Ad-EGFR-CD-533, as was the basal EGFR tyrosine phosphorylation (Fig. 1Go, C).

Previously, we demonstrated that MAPK is an important downstream target for radiation-induced EGFR activation. Inhibition of MAPK activation by the pharmacologic inhibitor PD98059 was associated with an abrogation of the radiation-induced cellular proliferative response (2,6). In addition, when MDA-EGFR-CD533 cells induced to express EGFR-CD533 were exposed to a single radiation dose of 2 Gy MAPK, activation was inhibited (6,13). To assess whether transduction of MDA-MB-231 with Ad-EGFR-CD533 could inhibit radiation-induced MAPK activation, we measured MAPK activity at multiple time points after irradiation with 2 Gy. We found that MDA-MB-231 cells transduced with Ad-EGFR-CD533 had a 77% reduction in the MAPK activity 5 minutes after irradiation (Fig. 1Go, D). Thus, expression of EGFR-CD533 inhibits the radiation-induced activation of MAPK.

EGFR-CD533 Expression and Radiosensitivity in Human Mammary Carcinoma Cells In Vitro

We next examined whether the transduction of MDA-MB-231 cells with Ad-EGFR-CD533 increased radiosensitivity. The radiosensitivity of MDA-MB-231 cells that were mock transduced or transduced with Ad-LacZ or Ad-EGFR-CD533 was determined in clonogenic survival assays. The survival curves yielded similar values for mock-transduced ( = 3.54; 95% CI = 3.33 to 3.74) and Ad-LacZ-transduced ( = 3.56; 95% CI = 3.29 to 3.84) MDA-MB-231 cells. By contrast, the value for MDA-MB-231 cells transduced with Ad-EGFR-CD533 was markedly reduced to 2.58 (95% CI = 2.38 to 2.79), which was a statistically significant increase in radiosensitivity (P<.001) when compared with the Ad-LacZ-transduced cells (DER = 1.39; 95% CI = 1.28 to 1.41) (Fig. 2Go, A).



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Fig. 2. Radiation dose–response analyses of MDA-MB-231 and MDA-EGFRCD533 cells under conditions of EGFR-CD533 overexpression. Clonogenic survival was determined after single radiation exposures between 1 and 8 Gy. Error bars represent the means and 95% confidence intervals from four independent experiments. A) Clonogenic survival of MDA-MB-231 cells that were mock transduced (MDA-MB-231) or transduced with an adenoviral vector containing the LacZ gene (MDA-Ad-LacZ) or Ad-EGFR-CD533 (MDA-Ad-EGFR-CD533), both at a multiplicity of infection of 50. B) Clonogenic survival of MDA-EGFR-CD533 cells lacking expression of EGFR-CD533 (i.e., without induction by doxycycline [–Dox]) or with expression of EGFR-CD533 (i.e., with induction by doxycycline [+Dox]). EGRF = epidermal growth factor receptor; DER = dose-enhanced ratio.

 
We have developed and reported on MDA-EGFR-CD533 cells that express EGFR-CD533 under the control of a doxycycline-inducible promoter (6). The radiosensitivity of this stably transfected MDA-EGFR-CD533 cell line after doxycycline-induced EGFR-CD533 expression was compared with that of MDA-MB-231 cells transduced with Ad-EGFR-CD533. MDA-EGFR-CD533 cells without induction of EGFR-CD533 (control cells) had a of 2.94 (95% CI = 2.81 to 3.06), which was similar to that of mock-transduced or Ad-LacZ-transduced MDA-MB-231 cells (see Fig. 2Go, A [MDA-MB-231 or MDA-AdLacZ] and B [–Dox]). MDA-EGFR-CD533 cells after doxycycline-induced EGFR-CD533 expression had a of 1.94 (95% CI = 2.19 to 1.69), which yielded a DER of 1.52 (95% CI = 1.34 to 1.74) and reflected a statistically significant increase in radiosensitivity relative to control cells (P<.001; Fig. 2Go, B). The radiosensitivity was similar for MDA-MB-231 cells transduced with Ad-EGFR-CD533 and MDA-EGFR-CD533 cells with doxycycline-induced EGFR-CD533 expression. Thus, these data demonstrate that EGFR-CD533 expression, whether from an adenoviral vector or from an inducible promoter in stable MDA-EGFR-CD533 cells, results in direct radiosensitization of MDA-MB-231 cells after single-dose radiation exposures.

Optimization of Intratumoral Infusion of Ad-EGFR-CD533 Into MDA-MB-231 Xenografts

For EGFR-CD533 to be considered an effective gene therapeutic agent, it was important to determine whether the mechanism of action in vivo was similar to that seen in vitro and whether the fraction of tumor cells transduced with Ad-EGFR-CD533 would influence the radiosensitivity of tumors. To establish optimal conditions for the administration of adenoviral vectors into MDA-MB-231 xenograft tumors, a fixed dose of Ad-LacZ (1010 pfu) was infused into tumors by use of the 4-, 6-, or 2 x 6-track infusion techniques. Transduction efficiencies, as assessed by {beta}-galactosidase positivity of cells after Ad-LacZ infusion, were 18% (95% CI = 12.6 to 25.8) and 24% (95% CI = 14.7 to 32.1) for the single 4- and 6-track infusions, respectively (Fig. 3Go, A). The transduction efficiency for Ad-LacZ after 2 x 6-track infusions with manual needle retraction was 44% (95% CI =38.4 to 52.6) (Fig. 3Go, A and B), which was statistically significantly higher (P = .012) than that after a single 6-track infusion.

Infusion of Ad-EGFR-CD533 with the 2 x 6-track technique (Fig. 3Go, B) resulted in levels of EGFR-CD533 protein expression that were similar to those of the endogenous wild-type EGFR (Fig. 3Go, C). The protein levels of both EGFR and EGFR-CD533 appeared to be substantially lower in cells isolated from MDA-MB-231 tumors than in MDA-MB-231 cells transduced with Ad-EGFR-CD533 in vitro, a finding that was noted previously for A-431 squamous carcinoma cells (34). In addition, tumors transduced with Ad-LacZ or Ad-EGFR-CD533 expressed higher levels of the wild-type and EGFR-CD533 protein after maintenance in vitro for 96 hours (Fig. 3Go, C; right panel). The results demonstrate that intratumoral administration of Ad-EGFR-CD533 can transduce tumor cells with effective transgene expression.

EGFR-CD533 Expression and Function in Human Mammary Carcinoma Cells In Vivo

We next examined the effects of expressing EGFR-CD533 in established MDA-MB-231 xenograft tumors. We determined radiation-induced EGFR activation in vivo and generated radiation dose–response data. Single radiation exposures of 4 Gy did not alter the level of EGFR protein but did induce activation of EGFR within 2–10 minutes, as detected by a 2.5-fold to fivefold increase in EGFR tyrosine phosphorylation (Fig. 4Go, A).



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Fig. 4. Radiation responses of epidermal growth factor receptor (EGFR) in vivo, expression and function of EGFR-CD533 in MDA-EGFR-CD533 xenograft tumors, and the effect of EGFR-CD533 expression in vivo on tumor radiosensitization. A) MDA-MB-231 tumors were exposed to a single radiation dose of 4 Gy. EGFR tyrosine phosphorylation (EGFR Tyr-P) was quantified by immunoblotting at the indicated time points after irradiation. The corresponding values of EGFR Tyr-P represent increases over the individual nonirradiated control (0 minutes). Blots were also probed for EGFR protein with an anti-EGFR monoclonal antibody (MAb) to demonstrate equal loading (right panel). The data are representative of three independent experiments. B) Left panel: EGFR-CD533 protein expression levels were determined in stably transfected MDA-EGFR-CD533 cells after doxycycline (Dox) treatment (1 µg/mL) in vivo and in vitro (see the "Materials and Methods" section). Right panel: MDA-EGFR-CD533 cells without (–Dox) and with (+Dox) doxycycline-induced expression of EGFR-CD533 in vivo were exposed to a single dose of 4 Gy. EGFR Tyr-P was quantified 10 minutes after radiation exposure. Blots were also probed for EGFR protein with an anti-EGFR MAb to demonstrate equal loading. The corresponding value of tyrosine phosphorylation represents the fold change relative to the control (–Dox). The data shown are representative of three independent experiments. C) The effect of EGFR-CD533 expression in vivo on the radiosensitivity of MDA-MB-231 and MDA-EGFR-CD533 tumors after irradiation with 1.5 Gy on 3 consecutive days by use of an ex vivo colony-formation assay. Left panel: Expression of LacZ or EGFR-CD533 was induced in MDA-MB-231 tumors after intratumoral infusion with 1010 plaque-forming units of adenoviral vector containing the bacterial LacZ gene (Ad-LacZ) or Ad-EGFR-CD533 by use of the 2 x 6-track infusion protocol on 2 consecutive days. Twenty-four hours after irradiation, the tumors were isolated, and single cells were plated in clonogenic survival assays. The surviving fraction of MDA-MB-231 tumor cells transduced with Ad-LacZ was set as 1.0. The data represent the means and 95% confidence intervals (CIs) of three independent experiments (six mice/group). Right panel: Expression of EGFR-CD533 was induced in MDA-EGFR-CD533 tumors after Dox treatment in vivo. The surviving fraction of MDA-EGFR-CD533 tumor cells without Dox treatment (–Dox) was set as 1.0. The data represent the means and 95% CIs of four independent experiments. DER = dose-enhancement ratio.

 
To determine the functional consequences of EGFR-CD533 expression on radiation-induced activation of EGFR in vivo, we used MDA-EGFR-CD533 tumors because all of the tumor cells could be induced to express EGFR-CD533 after intraperitoneal doxycycline administration. After establishing the conditions for maximal induction of EGFR-CD533 expression in MDA-EGFR-CD533 tumors by repeated intraperitoneal doxycycline injections, we compared the levels of maximum EGFR-CD533 expression in MDA-EGFR-CD533 tumors with those induced in vitro (Fig. 4Go, B). Of the 20 MDA-EGFR-CD533 tumors tested, greater than 80% expressed levels of EGFR-CD533 comparable to those induced in vitro, as determined by western analysis (Fig. 4Go, B), whereas the rest expressed intermediate or lower levels of the transgene (data not shown). Only tumors that expressed high levels of EGFR-CD533 were used for experiments to assess functional consequences of EGFR-CD533 expression. More important, radiation-induced EGFR activation was reduced by 2.94-fold (95% CI = 2.23 to 4.14) in doxycycline-induced MDA-EGFR-CD533 tumors (Fig. 3Go, B; right panel). These results demonstrate that radiation-induced EGFR activation occurred in tumors in vivo to similar extents as in cultured cells in vitro, and radiation-induced EGFR activation, both in vivo and in vitro, was completely inhibited by the expression of EGFR-CD533.

EGFR-CD533 Expression In Vivo and Tumor Radiosensitization

EGFR-CD533 radiosensitized MDA-MB-231 cells in vitro independent of the method of EGFR-CD533 induction. These studies were then extended to examine whether in vivo expression of EGFR-CD533 could radiosensitize MDA-MB-231 tumors with the use of ex vivo clonogenic survival assays after irradiation of the tumors. Because of our previous work on radiation-induced EGFR activation and proliferation responses during repeated radiation exposures (6,10,14), we elected to use a radiation protocol of three 1.5-Gy exposures for 3 consecutive days, which resulted in a 50% clonogenic survival compared with the unirradiated control tumors (data not shown). The radiosensitization of MDA-MB-231 cells after transduction with Ad-EGFR-CD533 was tested with the use of an optimized infusion and the same radiation schedule of three daily 1.5-Gy treatments (Fig. 3Go, B). Tumors infused with Ad-EGFR-CD533 had a statistically significant 46% reduction in clonogenic survival (P<.001), resulting in a DER of 1.85 (95% CI = 1.54 to 2.51) (Fig. 4Go, C) relative to Ad-LacZ vector controls.

The radiation response parameters of MDA-MB-231 tumors transduced with Ad-EGFR-CD533 were compared with MDA-EGFR-CD533 tumors in which EGFR-CD533 expression was induced in all tumor cells by repeated intraperitoneal doxycycline administration. Clonogenic survival data corrected for plating efficiencies indicated that MDA-EGFR-CD533 tumor cells, induced to express EGFR-CD533, had a statistically significant 38% reduction in clonogenic survival (P<.001) compared with control tumor cells, resulting in a DER of 1.61 (95% CI = 1.51 to 1.70) (Fig. 4Go, C).

Our results demonstrate that the degree of radiosensitization was comparable between MDA-MB-231 tumors, in which 44% of the cells were transduced with Ad-EGFR-CD533, and MDA-EGFR-CD533 tumors, in which all of the cells should express EGFR-CD533 after doxycycline induction. Furthermore, the results demonstrate the feasibility of using adenoviral vector-mediated gene therapy to achieve radiosensitization. The substantial radiosensitization seen after repeated radiation exposures adds to the potential clinical relevance of these studies.


    DISCUSSION
 Top
 Notes
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In this report, we have presented both in vitro and in vivo evidence to show that the disruption of EGFR function through the expression of a dominant-negative EGFR-CD533 resulted in a statistically significant radiosensitization after both single and repeated radiation exposures. We demonstrate that MDA-MB-231 xenografts could be transduced by Ad-EGFR-CD533 in vivo, which resulted in radiosensitization comparable to that seen with MDA-EGFR-CD533 tumors expressing EGFR-CD533 under control of a doxycycline-inducible promoter (6). Thus, the data from our preclinical xenograft model provide strong evidence that genetic disruption of EGFR function represents a potentially powerful tool for tumor cell radiosensitization.

The use of ex vivo experimentation allowed the in vivo effects to be quantified and the mechanisms of action previously established in vitro to be verified in vivo (5,6,13). Our results suggest that there are at least two mechanisms, previously linked to the expression of EGFR-CD533 (6,7), that could contribute to radiosensitization in vivo. One mechanism is the elimination of a radiation-induced proliferative response that we described with the use of MDA-EGFR-CD533 cells in a protocol of repeated radiation exposures (6,10). The other mechanism may relate to EGFR-CD533 potentially interfering with changes in cell cycle control and/or DNA repair, both of which could increase radiosensitivity in standard clonogenic survival experiments (35). Both direct radiosensitization and inhibition of cell proliferation can be expected to increase radiosensitivity in vivo (6).

We used clonogenic survival assays to assess in vivo radiosensitization because they allow a more direct comparison of cellular radiation response data with a number of other human tumor cell lines, such as squamous cell carcinoma and malignant glioma cells, in which the in vitro radiosensitization effects of EGFR-CD533 have been quantified by clonogenic survival analyses [Lammering G, Schmidt-Ullrich RK: unpublished data; (7)]. Another reason for preferring ex vivo colony formation over the alternative tumor growth-delay assays was our observation that the expression of EGFR-CD533 in vivo was associated with tumor growth retardation (Lammering G, Lin P-S, Schmidt-Ullrich RK: unpublished data). Because the growth delay of tumor xenografts is influenced by many factors other than tumor cell properties, such as expression of EGFR-CD533 in our experimental system, this assay would have seriously compromised our ability to quantify in vivo radiosensitization, one of the primary goals of this investigation. Given our focus on tumor cell radiosensitization and its underlying mechanisms related to EGFR function, ex vivo clonogenic survival assays established through the isolation of single tumor cells highly correlative data between cell survival and molecular responses (6,7,10,13).

Several other approaches to disable the ErbB family receptor function and to modulate tumor cell responses are being explored, including the use of a monoclonal antibody, C225, that binds to the EGF-ligand domain of EGFR, thereby preventing receptor signaling (3638), or the use of small molecule pharmacologic inhibition of the EGFR kinase domain (3941). Studies (37,38,4244) have demonstrated that the inhibition of EGFR or ErbB2 function sensitizes tumor cells to the toxic effects of ionizing radiation. In examining the underlying mechanisms of the EGFR blockade with C225 and the expression of EGFR-CD533, it is clear that both approaches inhibit EGFR function. There is, however, indirect evidence that the mechanism(s) of action of these two approaches is different. For example, increased expression of the cyclin-dependent kinase inhibitor p27Kip-1 may be part of the mechanism by which C225 reduces tumor cell growth and enhances radiosensitivity (37). By contrast, EGFR-CD533 decreases tumor cell growth and increases radiosensitivity through the inhibition of radiation-induced MAPK activation followed by increased p21Cip-1/WAF1 expression (45). Moreover, C225 and EGFR-CD533 target different functions of EGFR. Whereas both C225 and the tyrphostin AG1478 inhibit EGFR kinase activity, the former by promoting EGFR internalization and degradation (46) and the latter by binding directly to the EGFR catalytic cytoplasmic domain (47), EGFR-CD533 inhibits the activation of all ErbB receptors by preventing functional receptor heterodimerization and transphosphorylation (11,22,23). Thus, there may also be an additional therapeutic benefit for a combined treatment with EGFR-CD533 and other inhibitors of EGFR function.

Because overexpressed EGFR-CD533 interacts with all ErbB receptors, it may be a broadly active therapeutic tool applicable to a variety of tumor types. Both carcinomas and malignant gliomas exhibit great variation in relative expression levels of EGFR and other ErbB RTKs (48,49), which may substantially affect cellular responses to radiation (23). Furthermore, EGFR-CD533 may also prove to be useful in disrupting responses by other mutated EGFR species, such as EGFRvIII [(50,51); Lammering G, Hewit TH, Contessa JN, Schmidt-Ullrich RK: unpublished data]. The expression of constitutively active EGFRvIII, which lacks the EGF amino-terminal-binding domain (50), was first described for malignant gliomas (52,53) but is also found in carcinoma cell xenograft tumors (Lammering G, Hewit TH, Hawkins WT, Schmidt-Ullrich RK: unpublished data), affirming the notion that this receptor is expressed only in vivo (52,53). The functional consequences of EGFRvIII expression on cellular radiation responses are currently unknown and are being investigated in our laboratory.

In summary, our data provide evidence that EGFR function can be modulated effectively by a gene therapeutic approach of overexpressing a dominant-negative EGFR-CD533 by use of in vivo adenoviral vector delivery. This results in tumor cell radiosensitization in vitro and in vivo after single and repeated radiation exposures. The mechanisms underlying this radiosensitization involve disruption of major cytoprotective responses of radiation-induced proliferation and recovery capacity, mediated by radiation-induced activation of EGFR and MAPK. The tightly associated data from in vivo/in vitro experiments allowed us to demonstrate that the genetic disruption of EGFR function in autocrine growth-regulated tumor cells occurs by the same mechanisms in cultured cells as in xenograft tumors, thus affirming the therapeutic potential of this approach.


    NOTES
 
Supported by Public Health Service grants P01CA72955 and R01CA65896 (to R. Schmidt-Ullrich) from the National Cancer Institute, National Institutes of Health, Department of Health and Human Services; by the Deutsche Krebshilfe, Dr. Mildred Scheel Stiftung fellowship support (to G. Lammering); and by grant DAMD17–99–1-9426 (to Paul Dent) from the U.S. Army.

We thank Dr. Cyrus Amir from Departments of Radiation Oncology and Biostatistics, Medical College of Virginia Campus, Virginia Commonwealth University, Richmond, for his statistical analysis.


    REFERENCES
 Top
 Notes
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

1 Chryssogelos SA, Dickson RB. EGF receptor expression, regulation, and function in breast cancer. Breast Cancer Res Treat 1994;29:29–40.[Medline]

2 Ekstrand AJ, James CD, Cavenee WK, Seliger B, Pettersson RF, Collins VP. Genes for epidermal growth factor receptor, transforming growth factor and their expression in human gliomas in vivo. Cancer Res 1991;51:2164–72.[Abstract]

3 Alimandi M, Romano A, Curia MC, Muraro R, Fedi P, Aaronson SA, et al. Cooperative signaling of ErbB3 and ErbB2 in neoplastic transformation and human mammary carcinomas. Oncogene 1995;10:1813–21.[Medline]

4 Westphal M, Meima L, Szonyi E, Lofgren J, Meissner H, Hamel W, et al. Heregulins and the ErbB-2/3/4 receptors in gliomas. J Neurooncol 1997;35:335–46.[Medline]

5 Schmidt-Ullrich RK, Valerie K, Fogleman PB, Walters J. Radiation-induced autophosphorylation of epidermal growth factor receptor in human malignant mammary and squamous epithelial cells. Radiat Res 1996;145:81–5.[Medline]

6 Contessa JN, Reardon DB, Todd D, Dent P, Mikkelsen RB, Valerie K, et al. The inducible expression of dominant-negative epidermal growth factor receptor-CD533 results in radiosensitization of human mammary carcinoma cells. Clin Cancer Res 1999;5:405–11.[Abstract/Free Full Text]

7 Lammering G, Valerie K, Lin PS, Mikkelsen RB, Contessa JN, Feden JP, et al. Radiosensitization of malignant glioma cells through overexpression of dominant-negative epidermal growth factor receptor. Clin Cancer Res 2001;7:682–90.[Abstract/Free Full Text]

8 Schmidt-Ullrich RK, Valerie KC, Chan W, McWilliams D. Altered expression of epidermal growth factor receptor and estrogen receptor in MCF-7 cells after single and repeated radiation exposures. Int J Radiat Oncol Biol Phys 1994;29:813–9.[Medline]

9 Goldkorn T, Balaban N, Shannon M, Matsukuma K. EGF receptor phosphorylation is affected by ionizing radiation. Biochim Biophys Acta 1997;1358:289–99.[Medline]

10 Kavanagh BD, Lin PS, Chen P, Schmidt-Ullrich RK. Radiation-induced enhanced proliferation of human squamous cancer cells in vitro: a release from inhibition by epidermal growth factor. Clin Cancer Res 1995;1:1557–62.[Abstract]

11 Schmidt-Ullrich RK, Dent P, Grant S, Mikkelsen RB, Valerie K. Signal transduction and cellular radiation responses. Radiat Res 2000; 153:245–57.[Medline]

12 Carter S, Auer KL, Reardon DB, Birrer M, Fisher PB, Schmidt-Ullrich RK, et al. Inhibition of the mitogen activated protein (MAP) kinase cascade potentiates cell killing by low dose ionizing radiation in A431 human squamous carcinoma cells. Oncogene 1998;16:2787–96.[Medline]

13 Reardon DR, Contessa JN, Mikkelsen RB, Valerie K, Amir C, Dent P, et al. Dominant negative EGFR-CD533 and inhibition of MAPK modify JNK1 activation and enhance radiation toxicity of human mammary carcinoma cells. Oncogene 1999;18:4756–66.[Medline]

14 Schmidt-Ullrich RK, Mikkelsen RB, Dent P, Todd DG, Valerie K, Kavanagh BD, et al. Radiation-induced proliferation of the human A431 squamous carcinoma cells is dependent on EGFR tyrosine phosphorylation. Oncogene 1997;15:1191–7.[Medline]

15 Schmidt-Ullrich RK, Contessa JN, Dent P, Mikkelsen RB, Valerie K, Reardon DB, et al. Molecular mechanisms of radiation-induced accelerated repopulation. Radiat Oncol Investig 1999;7:321–30.[Medline]

16 Withers HR, Taylor JM, Maciejewski B. The hazard of accelerated tumor clonogen repopulation during radiotherapy. Acta Oncol 1988;27:131–46.[Medline]

17 Trott KR. The mechanisms of acceleration of repopulation in squamous epithelia during daily irradiation. Acta Oncol 1999;38:153–7.[Medline]

18 Tubiana M. Repopulation in human tumors. A biological background for fractionation in radiotherapy. Acta Oncol 1988;27:83–8.

19 Begg AC. Prediction of repopulation rates and radiosensitivity in human tumours. Int J Radiat Biol 1994;65:103–8.[Medline]

20 Fowler JF. Rapid repopulation in radiotherapy; a debate on mechanism. The phantom of tumor treatment—continually rapid proliferation unmasked. Radiother Oncol 1991;22:156–8.[Medline]

21 Redemann N, Holzmann B, von Rueden T, Wagner EF, Schlessinger J, Ullrich A. Anti-oncogenic activity of signaling-defective epidermal growth factor receptor mutants. Mol Cell Biol 1992;12:491–8.[Abstract]

22 Kashles O, Yarden Y, Fisher R, Ullrich A, Schlessinger J. A dominant negative mutation suppresses the function of normal epidermal growth factor receptors by heterodimerization. Mol Cell Biol 1991;11:1454–63.[Medline]

23 Bowers G, Reardon D, Hewit TH, Dent P, Mikkelsen RB, Valerie K, et al. The relative role of ErbB1–4 receptor tyrosine kinases in radiation signal transduction responses of human carcinoma cells. Oncogene 2001;20:1388–97.[Medline]

24 Valerie K. Viral vectors for gene delivery. In: Wu-Pong S, Rojanasakul Y, editors. Biopharmaceutical drug design and development. Clifton (NJ): Humana Press; 1999. p. 69–142.

25 Valerie K, Brust D, Farnsworth J, Amir C, Taher MM, Hershey C, et al. Improved radiosensitization of rat glioma cells with adenovirus-expressed mutant herpes simplex virus-thymidine kinase in combination with acyclovir. Cancer Gene Ther 2000;7:879–84.[Medline]

26 Brust D, Feden J, Farnsworth J, Amir C, Broaddus WC, Valerie K. Radiosensitization of rat glioma with bromodeoxycytidine and adenovirus expressing herpes simplex virus-thymidine kinase delivered by slow, rate-controlled positive pressure infusion. Cancer Gene Ther 2000;7:778–88.[Medline]

27 Ho KC, Lin PS. Safranin O counterstaining enhances the counting of {beta}-galactosidase-expressing cells. Biotechniques 1997;23:642.[Medline]

28 Prabhu SS, Broaddus WC, Gillies GT, Loudon WG, Chen ZJ, Smith B. Distribution of macromolecular dyes in brain using positive pressure infusion: a model for direct controlled delivery of therapeutic agents. Surg Neurol 1998;50:367–75; discussion 375.[Medline]

29 Dent P, Lavoinne A, Nakielny S, Caudwell FB, Watt P, Cohen P. The molecular mechanism by which insulin stimulates glycogen synthesis in mammalian skeletal muscle. Nature 1990;348:302–8.[Medline]

30 Hahn GM, Little JB. Plateau-phase cultures of mammalian cells: an in vitro model for human cancer. Curr Top Radiat Res Q 1972;8:39–83.[Medline]

31 Fertil B, Dertinger H, Courdi A, Malaise EP. Mean inactivation dose: a useful concept for intercomparison of human cell survival curves. Radiat Res 1984;99:73–84.[Medline]

32 Hall EJ. Cell survival curves. In: Hall EJ, Ryan JD, Cox K, Papadopoulos D, editors. Radiobiology for the radiologist. 4th ed. Philadelphia (PA): Lippincott; 1994. p. 41.

33 Brown JM, Lemmon MJ. Potentiation by the hypoxic cytotoxin SR 4233 of cell killing produced by fractionated irradiation of mouse tumor. Cancer Res 1990;50:7745–9.[Abstract]

34 Mansbridge JN, Ausserer WA, Knapp MA, Sutherland RM. Adaptation of EGF receptor signal transduction to three-dimensional culture conditions: changes in surface receptor expression and protein tyrosine phosphorylation. J Cell Physiol 1994;161:374–82.[Medline]

35 Khandelwal SR, Kavanagh BD, Lin PS, Truong QT, Lu J, Abraham DJ, et al. RSR13, an allosteric effector of haemoglobin, and carbogen radiosensitize FSAII and SCCVII tumours in C3H mice. Br J Cancer 1999;79:814–20.[Medline]

36 Park BW, Zhang HT, Wu C, Berezov A, Zhang X, Dua R, et al. Rationally designed anti-HER2/neu peptide mimetic disables P185HER2/neu tyrosine kinases in vitro and in vivo. Nat Biotechnol 2000;18:194–8.[Medline]

37 Huang SM, Harari PM. Modulation of radiation response after epidermal growth factor receptor blockade in squamous cell carcinomas: inhibition of damage repair, cell cycle kinetics, and tumor angiogenesis. Clin Cancer Res 2000;6:2166–74.[Abstract/Free Full Text]

38 Milas L, Mason K, Hunter N, Petersen S, Yamakawa M, Ang K, et al. In vivo enhancement of tumor radioresponse by C225 antiepidermal growth factor receptor antibody. Clin Cancer Res 2000;6:701–8.[Abstract/Free Full Text]

39 Lenferink AE, Simpson JF, Shawver LK, Coffey RJ, Forbes JT, Arteaga CL. Blockade of the epidermal growth factor receptor tyrosine kinase suppresses tumorigenesis in MMTV/Neu + MMTV/TGF-alpha bigenic mice. Proc Natl Acad Sci U S A 2000;97:9609–14.[Abstract/Free Full Text]

40 Peus D, Vasa RA, Meves A, Beyerle A, Pittelkow MR. UVB-induced epidermal growth factor receptor phosphorylation is critical for downstream signaling and keratinocyte survival. Photochem Photobiol 2000;72:135–40.[Medline]

41 Fan Z, Shang BY, Lu Y, Chou JL, Mendelsohn J. Reciprocal changes in p27(Kip1) and p21(Cip1) in growth inhibition mediated by blockade or overstimulation of epidermal growth factor receptors. Clin Cancer Res 1997;3:1943–8.[Abstract]

42 Bonner JA, Maihle NJ, Folven BR, Christianson TJ, Spain K. The interaction of epidermal growth factor and radiation in human head and neck squamous cell carcinoma cell lines with vastly different radiosensitivities. Int J Radiat Oncol Biol Phys 1994;29:243–7.[Medline]

43 Bos M, Mendelsohn J, Kim YM, Albanell J, Fry DW, Baselga J. PD153035, a tyrosine kinase inhibitor, prevents epidermal growth factor receptor activation and inhibits growth of cancer cells in a receptor number-dependent manner. Clin Cancer Res 1997;3:2099–106.[Abstract]

44 Baselga J, Norton L, Albanell J, Kim YM, Mendelsohn J. Recombinant humanized anti-HER2 antibody (Herceptin) enhances the antitumor activity of paclitaxel and doxorubicin against HER2/neu overexpressing human breast cancer xenografts. Cancer Res 1998;58:2825–31.[Abstract]

45 Park JS, Carter S, Reardon DB, Schmidt-Ullrich R, Dent P, Fisher PB. Roles for basal and stimulated p21 (Cip-1/WAF1/MDA6) expression and mitogen-activated protein kinase signaling in radiation-induced cell cycle checkpoint control in carcinoma cells. Mol Biol Cell 1999;10:4231–46.[Abstract/Free Full Text]

46 Prewett M, Rockwell P, Rockwell RF, Giorgio NA, Mendelsohn J, Scher HI, et al. The biologic effects of C225, a chimeric monoclonal antibody to the EGFR, on human prostate carcinoma. J Immunother Emphasis Tumor Immunol 1996;19:419–27.[Medline]

47 Levitzki A, Gazit A. Tyrosine kinase inhibition: an approach to drug development. Science 1995;267:1782–8.[Medline]

48 Alimandi M, Romano A, Curia MC, Muraro R, Fedi P, Aaronson SA, et al. Cooperative signaling of ErbB3 and ErbB2 in neoplastic transformation and human mammary carcinomas. Oncogene 1995;10:1813–21.[Medline]

49 Earp HS, Dawson TL, Li X, Yu H. Heterodimerization and functional interaction between EGF receptor family members: a new signaling paradigm with implications for breast cancer research. Breast Cancer Res Treat 1995;35:115–32.[Medline]

50 Batra SK, Castelino-Prabhu S, Wikstrand CJ, Zhu X, Humphrey PA, Friedman HS, et al. Epidermal growth factor ligand-independent, unregulated, cell-transforming potential of a naturally occurring human mutant EGFRvIII gene. Cell Growth Differ 1995;6:1251–9.[Abstract]

51 Moscatello DK, Holgado-Madruga M, Godwin AK, Ramirez G, Gunn G, Zoltick PW, et al. Frequent expression of a mutant growth factor receptor in multiple human tumors. Cancer Res 1995;55:5536–9.[Abstract]

52 Bigner SH, Humphrey PA, Wong AJ, Vogelstein B, Mark J, Friedman HS, et al. Characterization of the epidermal growth factor receptor in human glioma cell lines and xenografts. Cancer Res 1990;50:8017–22.[Abstract]

53 Nishikawa R, Ji XD, Harmon RC, Lazar CS, Gill GN, Cavenee WK, et al. A mutant epidermal growth factor receptor common in human glioma confers enhanced tumorigenicity. Proc Natl Acad Sci U S A 1994;91:7727–31.[Abstract]

Manuscript received November 15, 2000; revised April 6, 2001; accepted April 17, 2001.


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