[CANCER RESEARCH 59, 1935–1940, April 15, 1999]

Epidermal Growth Factor Receptor Blockade with C225 Modulates Proliferation,Apoptosis, and Radiosensitivity in Squamous Cell Carcinomas of theHead and Neck1

Shyh-Min Huang, Jonathan M. Bock, and Paul M. Harari2

Department of Human Oncology, University of Wisconsin School of Medicine and Comprehensive Cancer Center, Madison, Wisconsin 53792-0600

ABSTRACT

We examined effects of the anti-epidermal growth factor receptor mono-clonal antibody C225 on proliferation, cell cycle phase distribution, apoptosis,and radiosensitivity in squamous cell carcinoma (SCC) cell lines derived fromhead and neck cancer patients. Exposure to C225 in culture inhibits SCCproliferation in a time-dependent manner, and the degree of growth inhibi-tion, compared to controls, ranges from 20 to 75%. Flow cytometry analysisdemonstrates that C225 treatment induces accumulation of cells in G1, whichis accompanied by a 2–3-fold decrease in the percentage of cells in S phase.C225 exposure also induces apoptosis in SCC populations, as demonstratedby flow cytometry analysis using dual stainings of merocyanine 540 andHoechst 33342. Western blot analysis indicates that C225 exposure inducesaccumulation of hypophosphorylated retinoblastoma protein and increasesexpression of p27KIP1. An increase in Bax expression and concurrent decreasein Bcl-2 expression are observed when SCC cells are exposed to C225.Examination of C225 effects on radiation response in SCCs demonstratesenhancement in radiosensitivity and amplification of radiation-induced ap-optosis. These effects are observed in both single-dose and fractionated radi-ation experiments. C225 represents a promising growth-inhibitory agent thatcan influence cellular proliferation, apoptosis, and radiosensitivity in SCCs ofthe head and neck.

INTRODUCTION

SCCs3 of the H&N are among the most rapidly proliferating humansolid tumors, with median tumor potential doubling times of 3–5 daysmeasuredin vivo. Radiation therapy plays a primary role in treatmentfor patients with SCC of the H&N. However, the rapid proliferation ofsurviving tumor clonogens during a conventional 7–8-week course ofH&N radiotherapy serves to compromise ultimate tumor control (1).Intensified radiotherapy or chemotherapy regimens delivered over areduced treatment time represent one approach to counteract rapidtumor repopulation (2). Modest clinical gains have been demonstratedwith such treatment schedules, although they are generally accompa-nied by a prominent increase in acute toxicity (3). An alternativebiological approach, which uses antiproliferative agents such as theanti-EGFR mAb C225 to alter tumor proliferation and/or radiosensi-tivity, therefore, offers promise for improving tumor control (4).

The EGFR system represents a promising therapeutic target because itis commonly overexpressed in human epithelial tumors (5). Elevatedlevels of EGFR and its ligand, TGF-a, have been identified in themajority of primary tumors from patients with H&N SCCs (6). ThisEGFR/TGF-a overexpression has been shown to correlate with aggres-

sive malignant progression and poor clinical outcome (7, 8). In addition,a positive relationship between EGFR/TGF-a expression and prolifera-tion of surviving tumor cells after radiation therapy has been documented(9). Studies have reported that cell survival and repopulation may beregulated by the activation and expression of EGFR/TGF-a that isinduced following radiation (10, 11). In addition, the activation of down-stream effectors of the EGFR signaling pathway,i.e., ras andraf onco-genes, have been shown to increase cellular resistance to ionizing radi-ation (12). These findings suggest that EGFR blockade may be useful inreducing tumor cell repopulation by inhibition of tumor cell growthand/or modulation of cellular radiosensitivity.

C225 is a mouse-human chimeric anti-EGFR mAb. Blockade ofligand binding to EGFR by C225 inhibits activation of the receptortyrosine kinase (13). In addition, C225 has been shown to inhibit theproliferation of a variety of cultured malignant human cell lines thatoverexpress EGFR, including cells from vulva, breast, colon, lung,kidney, and prostate (14, 15). Inhibition of cell growth may resultfrom the induction of cell cycle arrest and/or induction of apoptosis(16, 17). In addition, treatment with C225 has been shown to augmentthe antitumor activity of several chemotherapeutic drugs in mousexenograft models (18). The objective of this study was to examine thepotential therapeutic utility of C225 in conjunction with radiation tocounteract tumor cell repopulation and to modulate cellular radiosen-sitivity and apoptosis in SCCs of the H&N.

MATERIALS AND METHODS

Chemicals and C225.Cell culture media were obtained from Life Tech-nologies, Inc. (Gaithersburg, MD). PI and Ho342 were obtained from Molec-ular Probes (Eugene, OR). Primary antibodies against Rb, p27KIP1, Bcl-2, andBax were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).Anti-a-tubulin antibody was obtained from Oncogene Research Products(Cambridge, MA). ECL chemiluminescence detection system was purchasedfrom Amersham (Arlington Heights, IL). All other chemicals were purchasedfrom Sigma Chemical Co. (St. Louis, MO). C225 was generously provided byImClone Systems Inc. (New York, NY).

Cell Lines and Cell Culture. Human SCC cell lines were established frombiopsies of H&N cancer patients. The SCC-13Y cell line was derived from thefacial epidermis and was provided by Dr. B. Lynn Allen-Hoffman (Universityof Wisconsin, Madison, WI). The SCC-1 (floor of mouth), SCC-11B (hypo-pharynx), and SCC-38 (tonsil) cell lines were provided by Dr. Thomas E.Carey (University of Michigan, Ann Arbor, MI). SCC cells were culturedroutinely in DMEM supplemented with 10% FC serum (Hyclone, Logan, UT),1 mg/ml hydrocortisone, 1% penicillin, and 1% streptomycin.

Growth Inhibition. The antiproliferative effect of C225 on thein vitrogrowth profile of each SCC cell line was examined following replicate platingof known numbers of single cells from each cell line into 100-mm culturedishes. After 24 h, 30 nM C225 was added to the medium; cells were thentrypsinized and counted with a hemacytometer at indicated time intervals.

Flow Cytometric Analysis. Control or C225-treated cells were harvestedby trypsinization, washed with PBS, and then fixed in 95% ethanol and storedat 4°C for up to 7 days prior to DNA analysis. After the removal of ethanol bycentrifugation, cells were then incubated with phosphate-citric acid buffer [0.2M Na2HPO4 (pH 7.8)-4 mM citric acid] at room temperature for 45 min. Aftercentrifugation, cells were then stained with a solution containing 33mg/ml PI,0.13 mg/ml RNase A, 10 mM EDTA, and 0.5% Triton X-100 at 4°C for 24 h.

Received 10/26/98; accepted 2/17/99.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby markedadvertisementin accordance with18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by National Cancer Institute Grant CA 66786 (to P. M. H.).2 To whom requests for reprints should be addressed, at Department of Human

Oncology, University of Wisconsin Comprehensive Cancer Center, 600 Highland Ave-nue, Madison, WI 53792-0600. Phone: (608) 263-8500; Fax: (608) 263-9167; E-mail:harari@humonc.wisc.edu.

3 The abbreviations used are: SCC, squamous cell carcinoma; H&N, head and neck;EGFR, epidermal growth factor receptor; mAb, monoclonal antibody; TGF-a, transform-ing growth factor-a; PI, propidium iodide; Ho342, Hoechst 33342; FC, Fetal Clone-II;MC540, merocyanine 540; CDK, cyclin-dependent kinase; EGF, epidermal growth factor;DNA-PK, DNA-dependent protein kinase.

1935

Research. on March 21, 2019. © 1999 American Association for Cancercancerres.aacrjournals.org Downloaded from

Stained nuclei were analyzed for DNA-PI fluorescence using a Becton Dick-inson FACScan flow cytometer. Resulting DNA distributions were analyzedby ModFit (Verity Software House Inc., Topsham, ME) for the proportion ofcells in apoptosis and in the G0/G1, S, and G2-M phases of the cell cycle.

Apoptosis Analysis. Apoptosis was evaluated following dual staining ofSCC cells with Ho342 and MC540, as described previously (19). Ho342 is aDNA-specific dye that allows for the analysis of cell cycle position, andMC540 detects membrane phospholipid changes that occur in apoptotic cells.Briefly, cells were harvested with 5 mM EDTA at 37°C. After centrifugation,cell pellets were resuspended in 900ml of PBS, followed by addition of 100ml of 50 mg/ml Ho342. Thereafter, cells were incubated for 30 min in the dark,pelleted, and resuspended in 100ml of PBS. Fourml of MC540 (1 mg/ml) wereadded, and cells were incubated for 20 min in the dark. Cells were pelleted,resuspended in 1 ml of PBS, and analyzed immediately by flow cytometry.

Immunoblotting Analysis. After treatment, cells were lysed with Tween20 lysis buffer [50 mM HEPES (pH 7.4), 150 mM NaCl, 0.1% Tween 20, 10%glycerol, 2.5 mM EGTA, 1 mM EDTA, 1 mM DTT, 1 mM phenylmethylsulfonylfluoride, 25 mg/ml leupeptin, and 25mg/ml aprotinin] and sonicated. Equalamounts of protein were analyzed by SDS-PAGE. Thereafter, proteins weretransferred to nitrocellulose membranes and analyzed by specific primaryantibodies against Rb, p27KIP1, Bcl-2, or Bax. Proteins were detected viaincubation with horseradish peroxidase-conjugated secondary antibodies andthe ECL chemiluminescence detection system.

Single-Dose and Fractionated Radiation Experiments.Exponentiallygrowing SCC cells in monolayer culture were irradiated in 100-mm Petridishes using a137Cs irradiator (J. L. Shepherd & Associates, Glendale, CA)with either a single radiation exposure (0, 3, 6, or 9 Gy) or repeated dailyexposures (0, 1, 2, or 3 Gy) for 3 consecutive days. After irradiation, cells wereharvested and replated for clonogenic survival analysis or flow cytometricanalysis to examine apoptosis. Single-dose and fractionated radiation experi-ments were carried out in control cells, cells exposed to C225 for 3 days beforeradiation (pretreatment), and cells exposed to C225 during and after radiationfor 3 days (posttreatment).

Radiation Survival. Survival following radiation exposure was defined as theability of the cells to maintain their clonogenic capacity and to form colonies.Briefly, after exposure to radiation, cells were trypsinized, counted, and seeded forcolony formation in 35-mm dishes at 50–5000 cells/dish. Following incubationintervals of 14–21 days, colonies were stained with crystal violet and manuallycounted. Colonies consisting of$50 cells were scored, and 4–10 replicate dishescontaining 10–150 colonies/dish were counted for each treatment. Differences inthe percentage of cell survivals among treatment groups were assessed by two-wayANOVA using generalized linear models in SAS.

RESULTS

C225 Inhibits SCC Proliferation. Growth curve profiles were eval-uated in each SCC cell line following the addition of 30 nM C225 directlyto the culture medium. These growth inhibition profiles over an 8-dayexposure period are depicted in Fig. 1. Exposure to C225 inhibitedcellular proliferation of SCC-1, SCC-11B, SCC-38, and SCC-13Y cellsin a time-dependent manner. Growth inhibition of SCC cell lines byC225, compared with that of untreated controls, ranged from 20 to 75%.

C225 Induces G0/G1 Arrest. The capacity of C225 to inhibit cellcycle progression was evaluated via flow cytometry. A representativeexample depicting the effect of C225 treatment on cell cycle phasedistribution in the SCC-13Y cell line is summarized in Fig. 2. Treat-ment with 30 nM C225 for 2 days resulted in the accumulation of cellsin the G0/G1 phase (64.6%) compared with that in controls (54.4%).A concurrent decrease in the percentage of cells in S phase wasobserved in C225-treated cells (8.0%) relative to that of the controls(23.1%). The G0/G1 accumulation and S-phase depletion were evenmore pronounced in cells treated with C225 for 4 days (70.5% and4.5%, respectively). No significant changes in the percentage of cellswithin G2-M phase were observed in these experiments.

To further characterize cell cycle events that might account for theobserved G0/G1 arrest, we examined the effect of C225 on theexpression of several key regulators of the G1-S phase transition. Asshown in Fig. 4, immunoblotting analysis showed that treatment with30 nM C225 increased the level of p27KIP1, which is an inhibitor of G1CDKs. In addition, treatment with C225 induced accumulation of thehypophosphorylated form of the Rb protein. The Rb protein is one ofthe critical substrates for G1 CDKs. Phosphorylation of Rb (hyper-phosphorylated Rb) and the subsequent release of transcription factorE2F are required for G1-S transition. Therefore, our results suggestthat treatment of SCC cells with C225 may induce G1 arrest byincreasing levels or activity of the CDK inhibitor, which results insubsequent accumulation of hypophosphorylated Rb.

Fig. 1. C225 inhibits proliferation of human SCC cellsin vitro. Known numbers ofsingle cells from SCC-13Y, SCC-38, SCC-1, or SCC-11B were plated into 100-mmculture Petri dishes in DMEM containing 10% FC serum. After 24 h, cells were treatedwith (F) or without (M) 30 nM C225. Cells were then harvested by trypsinization andcounted by hemacytometer with trypan blue dye exclusion at indicated time intervals.Data points, means of duplicate samples;bars, SD.

Fig. 2. C225 induces accumulation of cells in G1 with accompanying decrease in cellswithin S phase. SCC-13Y cells were harvested following a 2- or 4-day exposure to C225(30 nM). Cells were fixed and stained with PI for flow cytometry analysis as described in“Materials and Methods.” DNA histograms were modeled with ModFit analysis software,and phase percentages for G0/G1, S, and G2-M are depicted.Columns, mean values ofduplicate samples.

1936

C225 MODULATION OF PROLIFERATION AND RADIATION RESPONSE

Research. on March 21, 2019. © 1999 American Association for Cancercancerres.aacrjournals.org Downloaded from

C225 Induces Apoptosis.To determine whether C225 influ-enced SCC apoptotic response, we exposed SCC-13Y cells to 30nM C225 for various intervals and analyzed for apoptosis by flowcytometry using Ho342 and MC540 staining. As described previ-ously, Ho342 is a DNA-specific dye that allows for analysis of cellcycle position and MC540 detects membrane phospholipid changeswhich occur in apoptotic cells. Dual staining of Ho342 and MC540enables the identification of five distinct subpopulations, R2–R6.Subpopulations R2 and R3 represent viable cells, R4 and R5represent early apoptotic cells, and R6 represents cells undergoingthe latter stages of apoptosis by fragmenting their DNA. As shownin Fig. 3A, exposure of SCC-13Y to 30 nM C225 gradually in-creased cell numbers within the R4 –R6 subpopulations with aconcurrent decrease in the viable cell populations (R2 and R3).More specifically, a significant accumulation of cells (6 –7-fold) inthe R6 subpopulation was observed when cells were exposed to

C225 for 72 h (Fig. 3B). In contrast, no significant changes in thedistribution of cells among the five subpopulations were observedin control groups over a 72-h evaluation. These results suggest thattreatment of SCC-13Y cells with C225 not only induces G0/G1 cellcycle arrest but also induces apoptosis.

To further investigate the induction of apoptosis by C225, weconducted immunoblotting analysis to examine the expression ofBcl-2 and Bax, which are the critical regulators of apoptosis. Ingeneral, it is known that Bcl-2 protects cells from apoptosis, whereasBax promotes apoptosis. The propensity of a cell to undergo apoptosisis dependent in part on the Bax:Bcl-2 ratio within the cell (20). Asshown in Fig. 4, treatment of SCC-13Y cells with C225 reduced Bcl-2expression and increased Bax expression. Consistent with the previ-ous analysis of apoptosis using Ho342/MC540 staining, these resultssupport the finding that exposure of SCC-13Y to C225 increases theBax:Bcl-2 ratio and promotes the progression of cells to apoptosis.

Fig. 3. C225 induces apoptosis. Apoptosis was as-sessed by dual staining of SCC-13Y cells with Ho342and MC540 and analyzed by flow cytometry as de-scribed in “Materials and Methods.”A, dual-parameterdot plots of Ho342 staining (X axis) versusMC540binding (Y axis) of control and C225 (30 nM) treatedcells at indicated time intervals.Top left, five distinctsubpopulations of cells (R2–R6) are identified withrepresentative gating. The five subpopulations are de-fined as follows: R2, cells with 2 N DNA that areMC540 negative/dull; R3, cells with greater than 2 NDNA that are MC540 negative/dull; R4, cells with 2 NDNA that are MC540 bright; R5, cells with greater than2 N DNA that are MC540 bright; R6, cells with reducedHo342 staining (fragmented DNA) and MC540 bright.B, 6–7-fold increase in the fragmented DNA subgroupR6 (corresponding to latter phase of apoptosis) withlonger exposure duration to C225.

1937

C225 MODULATION OF PROLIFERATION AND RADIATION RESPONSE

Research. on March 21, 2019. © 1999 American Association for Cancercancerres.aacrjournals.org Downloaded from

C225 Enhances Radiosensitivity.To examine the potential use-fulness of combining C225 with radiation therapy for rapidly dividinghuman SCCs of the H&N, we conducted experiments to determine theeffects of C225 on single-dose and fractionated radiation on thesurvival of SCC-13Y cells. Fig. 5 depicts radiation-survival curves forcells exposed to C225 either before or after exposure to radiation.Exposure of SCC-13Y cells to C225 alone (30 nM) induced a reduc-tion in plating efficiency of;15–20%. On a single-dose radiationschedule (Fig. 5,left), both preirradiation and postirradiation treat-ments with C225 increased the radiation cell kill. Exposure to C225following radiation appeared to be slightly more effective in reducingcell survival compared with preradiation treatment by C225. Simi-larly, the effect of C225 on clonogenic survival was investigated usinga fractionated radiation treatment schedule (Fig. 5,right). In both thepreradiation and postradiation exposure settings, treatment with C225reduced cell survival significantly (P 5 0.001, two-way ANOVA).

C225 Enhances Radiation-induced Apoptosis.To further exam-ine the capacity of C225 to influence radiation-induced apoptosis, weharvested control and irradiated SCC-13Y cells and replated them in

DMEM with 10% FC serum. After 3 days of incubation, cells wereanalyzed for sub-G0 apoptotic population by flow cytometric analysis,as described previously. Fig. 6A demonstrates these results followingsingle-dose radiation exposure. Radiation alone (6 Gy) produced a2-fold induction of apoptosis, whereas exposure to 30 nM C225 incombination with 6 Gy of radiation induced apoptosis 5–6-fold,compared to untreated controls. Treatment with 30 nM C225 aloneinduced apoptosis by;2-fold. Similar results were found using thefractionated radiation treatment schedules (Fig. 6B).

DISCUSSION

The proliferation of tumor cells during radiation treatment has beenidentified as a factor that adversely impacts tumor response andultimate local control (1–3). One approach to reduce the impact oftumor cell repopulation during treatment involves the delivery ofbiological agents that slow or inhibit tumor cell proliferation. Here,we investigated the capacity of the anti-EGFR mAb C225 to inhibitproliferative growth and also examined the capacity of C225 tomodulate radiosensitivity in SCCs of the H&N.

Our results demonstrate that C225 inhibits growth across a varietyof SCC cell lines (all cell lines tested to date). The extent of growthinhibition ranges from 20 to 75% (Fig. 1). Growth inhibition of SCCsby C225 is accompanied by an accumulation of cells in G1 and by theinduction of apoptosis (Figs. 2 and 3). The G1 arrest correlates with anincrease in the level of the cell cycle inhibitor p27KIP, which is knownto inactivate G1 CDKs (Fig. 4). We also observed that exposure ofSCCs to C225 stimulates production of hypophosphorylated Rb, anincrease in the expression of Bax protein, and a decrease in theexpression of Bcl-2 (Fig. 4). These results are consistent with previousreports using a variety of other human tumor cell lines (16, 17, 21).

Many components of the biological response to ionizing radiationin mammalian cells are mediated through signal transduction, cellcycle regulation, and DNA repair pathways (22). Growth factors playan important role in modulating these cellular responses to radiation.Although thein vitro effects of EGF on the radiosensitivity of tumorcells have been extensively studied, inconsistent results have beendemonstrated. For example, Wollmanet al. (23) showed that preirra-diation exposure to EGF enhanced radioresistance in MCF-7 cells. Incontrast, Kwok and Sutherland (24) reported that the presence of EGFbefore, during, or after irradiation enhanced radiosensitivity in CaSkicells. Additional studies demonstrated that, whereas some squamouscancer cell lines are sensitized by EGF (e.g.,A431, SCC-6, and HN5),

Fig. 4. C225 exposure modulates expression of proteins involved in cell cycle regu-lation and apoptosis. Exponentially growing cells were treated with C225 (30 nM) forvarious time intervals and harvested. Following cell lysis, the lysates were processed forimmunoblotting using antibodies directed against Rb, p27KIP1, Bcl-2, and Bax as de-scribed in “Materials and Methods.”Arrows, hyper- and hypophosphorylated forms of Rb(hyperRbandhypoRb, respectively).a-Tubulin serves as a loading control.

Fig. 5. C225 enhances radiosensitivity. The influence of 30 nM

C225 on intrinsic radiosensitivity was examined by clonogenic sur-vival in SCC-13Y cells following exposure to single dose (left) orfractionated radiation (right) as described in “Materials and Methods.”For single-dose experiments, cells were exposed to C225 for 3 daysbefore irradiation (pretreatment) or exposed to C225 during and afterirradiation (posttreatment). For fractionated experiments, cells wereexposed to C225 for 3 days followed by daily irradiation at 0, 1, 2, or3 Gy for 3 consecutive days (pretreatment) or were irradiated in thepresence of C225 for 3 days (posttreatment). Control samples are cellsexposed to radiation without C225 treatment.Data points, means ofduplicates with five cultures per replicate per point;bars, SD.

1938

C225 MODULATION OF PROLIFERATION AND RADIATION RESPONSE

Research. on March 21, 2019. © 1999 American Association for Cancercancerres.aacrjournals.org Downloaded from

others are unaffected by the presence of EGF following radiation (e.g.,SiHa and SCC-1; Refs. 25 and 26). One hypothesis regarding theaforementioned differential responses to EGF across various cancercell lines is that cells that are growth stimulated by EGF are radio-protected, whereas those that are growth inhibited by EGF are radio-sensitized (23). However, the underlying mechanisms remain to bedetermined.

Our results indicate that exposure to C225, either before or follow-ing radiation, enhanced the radiosensitivity of SCC-13Y cells (Fig. 5).This finding is reproducibly observed with both single-dose and

fractionated radiation treatment schemes. C225 exposure also en-hanced radiosensitivity in SCC-38 cells under the same experimentalconditions (data not shown). It is possible that the enhancement ofradiosensitivity by C225 is mediated by mechanisms involving DNArepair inhibition. Such speculation derives from recent findings byBandyopadhyayet al. (27), who demonstrated that C225 but not EGFtriggers a specific physical interaction between internalized EGFRand DNA-PK in the cytosol in a variety of cell types. Significantreduction in the level and activity of DNA-PK in the nucleus withconcurrent increase in DNA-PK levels in the cytosol were observed.

Fig. 6. C225 enhances radiation-induced apop-tosis. Control or C225 treated SCC-13Y cells wereeither irradiated with single doses of 6 Gy (A) orirradiated daily at 2 Gy for 3 consecutive days (B).Following radiation, cells were replated, incubatedfor 3 days and processed for flow cytometric anal-ysis as described in “Materials and Methods.” DNAhistograms of cells either untreated (Control),treated with C225 prior to irradiation (Pretreat-ment), or treated with C225 after irradiation (Post-treatment) are shown. Percentages of apoptosiswere determined on the basis of sub-G0 DNA con-tent (M1) of the histogram.

1939

C225 MODULATION OF PROLIFERATION AND RADIATION RESPONSE

Research. on March 21, 2019. © 1999 American Association for Cancercancerres.aacrjournals.org Downloaded from

Because DNA-PK is believed to play a major role in repairing DNAdouble-strand breaks, these findings suggest that C225 may impairDNA repair by reducing the nuclear level of DNA-PK.

The capacity of C225 to modulate tumor cell proliferation and cellcycle phase distribution may also play a central role regarding theobserved enhancement of radiosensitivity. Prior studies have suggestedthat modulation of radiosensitivity by EGF is cell cycle dependent, withcells in G1 phase being more radioresponsive and cells in the S phasebeing more radioresistant (28, 29). These results suggest that C225 mayenhance radiosensitivity by inducing accumulation of cells in a moreradiosensitive cell cycle phase (G1) and reducing the size of the radiore-sistant S-phase fraction. Another underlying mechanism that may con-tribute to the influence of C225 on radiosensitivity involves the avail-ability and activity of free radical scavengers such as glutathione. Priorwork in MCF-7 cells has identified that preradiation exposure to C225enhanced radiosensitivity, which was accompanied by a decrease inglutathione levels and the fraction of cells within S phase (23).

The success of radiation therapy in effecting local tumor controldepends on several factors, including overall clonogenic burden,intrinsic radiosensitivity, oxygenation status, repopulation capacity,and repair. Apoptosis may also affect the ultimate tumor response toradiation by influencing the overall clonogenic burden through pro-motion of cell loss during treatment (30, 31). In tumor cells withapoptotic propensity, death may be triggered by lower radiation dosesthan that required to induce reproductive cell death. Our resultsindicate that treatment with C225 not only enhances reproductive celldeath following radiation but also increases the fraction of tumor cellssuccumbing to radiation-induced apoptosis (Fig. 6).

In conclusion, findings from the present study indicate that C225is an effective antiproliferative agent for SCCs of the H&N.Growth inhibition of SCC cells is mediated through the inductionof G1 cell cycle arrest and the induction of apoptosis. In addition,we have confirmed that exposure of human SCCs to C225 en-hances the effects of radiation therapy. Specifically, exposure toC225 enhances reproductive cell death and radiation-induced ap-optosis in SCCs using both single-dose and fractionated radiationtreatment schedules. Several biological and clinical characteristicsof human SCCs of the H&N appear to lend themselves particularlywell to the investigation of strategies that involve EGFR blockadeplus radiation: (a) high EGFR expression levels, (b) rapid prolif-erative capacity, (c) radiation as a dominant treatment modality,and (d) locoregional recurrence as the dominant failure pattern.These characteristics in combination with the current findingssuggest C225 as a promising agent for further investigation in thetreatment of rapidly proliferating SCCs of the H&N. The potentialtherapeutic benefits of combining C225 with radiation therapy forpatients with advanced SCCs of the H&N is scheduled to com-mence Phase III clinical trial evaluation in 1999.

ACKNOWLEDGMENTS

We thank Kathleen Schell and Kristin Elmer for their assistance in the flowcytometry facility at the University of Wisconsin Comprehensive CancerCenter. We also wish to thank ImClone Systems Incorporated for kindlyproviding us with C225.

REFERENCES

1. Fowler, J. F., and Lindstrom, M. J. Loss of local control with prolongation inradiotherapy. Int. J. Radiat. Oncol. Biol. Phys.,23: 457–467, 1992.

2. Harari, P. M., and Kinsella, T. J. Advances in radiation therapy for head and neckcancer. Curr. Opin. Oncol.,7: 248–254, 1995.

3. McGinn, C. J., Harari, P. M., Fowler, J. F., Ford, C. N., Pyle, G. M., and Kinsella,T. J. Dose intensification in curative head and neck cancer radiotherapy–linear

quadratic analysis and preliminary assessment of clinical results. Int. J. Radiat. Oncol.Biol. Phys.,27: 363–369, 1993.

4. Huang, S., and Harari, P. M. Effects of anti-epidermal growth factor receptor monoclonalantibody 225 on proliferation, apoptosis and radiosensitivity in human squamous cellcarcinomas of the head and neck. Proc. Am. Assoc. Cancer Res.,39: 64, 1998.

5. Khazaie, K., Schirrmacher, V., and Lichtner, R. B. EGF receptor in neoplasia andmetastasis. Cancer Metastasis Rev.,12: 255–274, 1993.

6. Grandis, J. R., and Tweardy, D. J. Elevated levels of transforming growth factora andepidermal growth factor receptor messenger RNA are early markers of carcinogenesisin head and neck cancer. Cancer Res.,53: 3579–3584, 1993.

7. Grandis, J. R., Melhem, M. F., Gooding, W. E., Day, R., Holst, V. A., Wagener,M. M., Drenning, S. D., and Tweardy, D. J. Levels of TGF-a and EGFR protein inhead and neck squamous cell carcinoma and patient survival. J. Natl. Cancer Inst.(Bethesda),90: 824–832, 1998.

8. Maurizi, M., Almadori, G., Ferrandina, G., Distefano, M., Romanini, M. E., Cadoni,G., Benedetti-Panici, P., Paludetti, G., Scambia, G., and Mancuso, S. Prognosticsignificance of epidermal growth factor receptor in laryngeal squamous cell carci-noma. Br. J. Cancer,74: 1253–1257, 1996.

9. Peter, R. U., Beetz, A., Ried, C., Michel, G., van Beuningen, D., and Ruzicka, T.Increased expression of the epidermal growth factor receptor in human epidermalkeratinocytes after exposure to ionizing radiation. Radiat. Res.,136: 65–70, 1993.

10. Schmidt-Ullrich, R. K., Valerie, K. C., Chan, W., and McWilliams, D. Alteredexpression of epidermal growth factor receptor and estrogen receptor in MCF-7 cellsafter single and repeated radiation exposures. Int. J. Radiat. Oncol. Biol. Phys.,29:813–819, 1994.

11. Schmidt-Ullrich, R. K., Mikkelsen, R. B., Dent, P., Todd, D. G., Valerie, K.,Kavanagh, B. D., Contessa, J. N., Rorrer, W. K., and Chen, P. B. Radiation-inducedproliferation of the human A431 squamous carcinoma cells is dependent on EGFRtyrosine phosphorylation. Oncogene,15: 1191–1197, 1997.

12. Kasid, U., Pirollo, K., Dritschilo, A., and Chang, E. Oncogenic basis of radiationresistance. Adv. Cancer Res.,61: 195–233, 1993.

13. Fan, Z., Masui, H., Altas, I., and Mendelsohn, J. Blockade of epidermal growth factorreceptor function by bivalent and monovalent fragments of 225 anti-epidermal growthfactor receptor monoclonal antibodies. Cancer Res.,53: 4322–4328, 1993.

14. Fan, Z., Lu, Y., Wu, X., and Mendelsohn, J. Antibody-induced epidermal growthfactor receptor dimerization mediates inhibition of autocrine proliferation of A431squamous carcinoma cells. J. Biol. Chem.,269: 27595–27602, 1994.

15. Prewett, M., Rockwell, P., Rockwell, R. F., Giorgio, N. A., Mendelsohn, J., Scher, H. I.,and Goldstein, N. I. The biologic effects of C225, a chimeric monoclonal antibody to theEGFR, on human prostate carcinoma. J. Immunother.,19: 419–427, 1996.

16. Wu, X., Fan, Z., Masui, H., Rosen, N., and Mendelsohn, J. Apoptosis induced by ananti-epidermal growth factor receptor monoclonal antibody in a human colorectalcarcinoma cell line and its delay by insulin. J. Clin. Invest.,95: 1897–1905, 1995.

17. Wu, X., Rubin, M., Fan, Z., DeBlasio, T., Soos, T., Koff, A., and Mendelsohn,J. Involvement of p27KIP1 in G1 arrest mediated by an anti-epidermal growth factorreceptor monoclonal antibody. Oncogene,12: 1397–1403, 1996.

18. Mendelsohn, J., and Fan, Z. Epidermal growth factor receptor family and chemosen-sitization. J. Natl. Cancer Inst. (Bethesda),89: 341–343, 1997.

19. Reid, S., Cross, R., and Snow, E. C. Combined Hoechst 33342 and merocyanine 540staining to examine murine B cell cycle stage, viability and apoptosis. J. Immunol.Methods,192: 43–54, 1996.

20. Pepper, C., Hoy, T., and Bentley, D. P. Bcl-2/Bax ratios in chronic lymphocyticleukaemia and their correlation within vitro apoptosis and clinical resistance. Br. J.Cancer,76: 935–938, 1997.

21. Peng, D., Fan, Z., Lu, Y., DeBlasio, T., Scher, H., and Mendelsohn, J. Anti-epidermalgrowth factor receptor monoclonal antibody 225 up-regulates p27KIP1 and induces G1arrest in prostatic cancer cell line DU145. Cancer Res.,56: 3666–3669, 1996.

22. Coleman, C. N., and Stevenson, M. A. Biologic basis for radiation oncology.Oncology,10: 399–411, 1996.

23. Wollman, R., Yahalom, J., Maxy, R., Pinto, J., and Fuks, Z. Effect of epidermalgrowth factor on the growth and radiation sensitivity of human breast cancer cellsinvitro. Int. J. Radiat. Oncol. Biol. Phys.,30: 91–98, 1994.

24. Kwok, T. T., and Sutherland, R. M. Enhancement of sensitivity of human squamouscarcinoma cells to radiation by epidermal growth factor. J. Natl. Cancer Inst. (Be-thesda),81: 1020–1024, 1989.

25. Kwok, T. T., and Sutherland, R. M. Differences in EGF related radiosensitisation ofhuman squamous carcinoma cells with high and low numbers of EGF receptors. Br. J.Cancer,64: 251–254, 1991.

26. Bonner, J. A., Maihle, N. J., Folven, B. R., Christianson, T. J., and Spain, K. Theinteraction of epidermal growth factor and radiation in human head and neck squa-mous cell carcinoma cell lines with vastly different radiosensitivities. Int. J. Radiat.Oncol. Biol. Phys.,29: 243–247, 1994.

27. Bandyopadhyay, D., Mandal, M., Adam, L., Mendelsohn, J., and Kumar, R. Physicalinteraction between epidermal growth factor receptor and DNA-dependent proteinkinase in mammalian cells. J. Biol. Chem.,273: 1568–1573, 1998.

28. Kwok, T. T., and Sutherland, R. M. Cell cycle dependence of epidermal growth factorinduced radiosensitization. Int. J. Radiat. Oncol. Biol. Phys.,22: 525–527, 1992.

29. Laderoute, K. R., Ausserer, W. A., Knapp, A. M., Grant, T. D., and Sutherland, R. M.Epidermal growth factor modifies cell cycle control in A431 human squamouscarcinoma cells damaged by ionizing radiation. Cancer Res.,54: 1407–1411, 1994.

30. Meyn, R. E. Apoptosis and response to radiation: implications for radiation therapy.Oncology,11: 349–356, 1997.

31. Dewey, W. C., Ling, C. C., and Meyn, R. E. Radiation-induced apoptosis: relevanceto radiotherapy. Int. J. Radiat. Oncol. Biol. Phys.,33: 781–796, 1995.

1940

C225 MODULATION OF PROLIFERATION AND RADIATION RESPONSE

Research. on March 21, 2019. © 1999 American Association for Cancercancerres.aacrjournals.org Downloaded from

1999;59:1935-1940. Cancer Res   Shyh-Min Huang, Jonathan M. Bock and Paul M. Harari  Squamous Cell Carcinomas of the Head and NeckModulates Proliferation, Apoptosis, and Radiosensitivity in Epidermal Growth Factor Receptor Blockade with C225

  Updated version

  http://cancerres.aacrjournals.org/content/59/8/1935

Access the most recent version of this article at:

   

   

  Cited articles

  http://cancerres.aacrjournals.org/content/59/8/1935.full#ref-list-1

This article cites 30 articles, 6 of which you can access for free at:

  Citing articles

  http://cancerres.aacrjournals.org/content/59/8/1935.full#related-urls

This article has been cited by 84 HighWire-hosted articles. Access the articles at:

   

  E-mail alerts related to this article or journal.Sign up to receive free email-alerts

  Subscriptions

Reprints and

  .pubs@aacr.orgDepartment at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

  Permissions

  Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://cancerres.aacrjournals.org/content/59/8/1935To request permission to re-use all or part of this article, use this link

Research. on March 21, 2019. © 1999 American Association for Cancercancerres.aacrjournals.org Downloaded from