(CANCER RESEARCH 51. 1294-1299. February 15, 1991]

Upregulation of Epidermal Growth Factor Receptor Induced by a-Interferon inHuman Epidermoid Cancer Cells'

Alfredo Budillon,2 Pierosandro Tagliaferri, Michele Caraglia, Maria Rosaria Torrisi, Nicola Normanno,

Stefano lacobelli, Giovannella Palmieri, Maria Patrizia Stoppelli, Luigi Frati, and Angelo Raffaele BiancoCattedra di Oncologia Medica, lìFacoltàdi Medicina, Universitàdi Napoli, Napoli [A. B., P. T., M. C., M. A'., G. P., A. R. B.J; Dipartimento di Medicina Sperimentale,Università La Sapienza, Roma ¡M.R. T., L. F./; Cattedra di Oncologia Clínica,Università G. D'Annunzio, Chieti [S. I.]; and Istituto Internazionale di Genetica e

Biofisica, Napoli ¡M.P. S.]; Italy

ABSTRACT

Unregulated or increased expression of epidermal growth factor receptor (EGF-R) is a common event in neoplastic transformation; modulationof such a receptor by physiological agents could be, therefore, of clinicalinterest. We have studied the binding ability, the availability at cellsurface, and the synthesis of EGF-R in the A431 and KB human epider-moid cancer cell lines after treatment with recombinant a-interferon(IFN-a). After 48 h of treatment, IFN-a induces, in both cell lines,growth inhibition and enhances class I major histocompatibility HLAcomplex expression, which is a common marker of IFN action. [I25I]EGFtotal binding assessed after 48 h of treatment with IFN-a shows a dose-dependent upregulation of EGF-R binding capacity. Saturation plots ofthe binding data show that IFN-a treatment does not dramatically alterthe affinity of the EGF-R and indicate that IFN-a only increases thenumber of low affinity receptors. We show that this effect is due to aspecific increase in the synthesis of the receptor protein, as assessed byimmunoprecipitation of |"S]methionine-labeled cell extracts. Electronmicroscopy analysis has confirmed an increase of EGF-R proteins at cellsurface without major changes in the morphology of the cells. Takentogether, these results indicate that IFN-a consistently induces both thebinding capacity and the synthesis of EGF-R in human epidermoid cancercells and suggest the use of such a mechanism for new anticancertherapies.

INTRODUCTION

EGF1 is a potent mitogen for epidermal tissues in vivo and

induces proliferation of many cultured cell lines (1). EGF exertsits effects through the binding to a specific cell surface receptor.The EGF-R is a glycoprotein with a molecular weight of170,000, with intrinsic tyrosine-specific protein kinase activity(2) which shows homologies to \-erbB oncogene (3) and isconsidered the cellular effector of transforming growth factor-

a (4). In primary squamous cell carcinomas, it has been frequently found, relatively to normal tissues, an overexpressionof EGF-R kinase and of c-erbB mRNA, often due to geneamplification (5-7). EGF-R expression is also considered anunfavorable prognostic factor in human breast cancer (8). Overexpression of EGF-R could be therefore considered a specificmarker of cell transformation and could be used as a target foranticancer therapeutic approaches involving the use of monoclonal antibodies or hormone analogues (9, 10). EGF-R, therefore, behaves as a tumor-associated antigen and has a definedrole in the control to tumor cell growth. In this view pharma-

Received 8/9/90; accepted 11/30/90.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1Supported by the Italian Association for Cancer Research (AIRC).2To whom requests for reprints should be addressed, al Cattedra di Oncologia

Medica, II Facoltà di Medicina. Università di Napoli, via S. Pansini 5. 80131.Napoli, Italy.

1The abbreviations used are: EGF. epidermal growth faeton EGF-R. epidermalgrowth factor receptor; IFN-a. a-interferon; BSA, bovine serum albumin: FBS.fetal bovine serum: PBS. phosphate-buffered saline: DMEM. Dulbecco's modifiedEagle's medium: HEPES. jV-2-hydroxyethyïpiperazine-/V-2'-ethanesulfonic acid:

MAb. monoclonal antibody.

cological modulation of such a receptor by the use of exogenousnoncytotoxic agents, could be of major clinical interest (lilo).

It has been recently shown that IFN-a, in addition to itsstrong antiproliferative effect, is able to induce several changesin the tumor cell phenotype and to enhance the expression ofclass I HLA as well as of tumor-associated antigens, in humancancer cultured cells (17-19). On the other hand, availability ofrecombinant IFN-a for clinical use has paralleled its increasinguse in the treatment of human cancer; however, the biologicalbases of its antitumor activity remain mostly undefined and itstrue potential is probably underemphasized (20).

The aim of this study is to define the effects of IFN-a on thebinding ability, the availability at cell surface, and the expression level of EGF-R in A431 and KB human epidermoid cancercell lines, which express a large number of EGF-Rs at cellsurface and are growth inhibited by IFN-a. It must be pointedout that the two cell lines respond differently to epidermalgrowth factor: while A431 are growth inhibited (21), KB cellsare growth stimulated by EGF (22).

Understanding of the cellular mechanisms involved in EGF-R modulation by IFN-«may provide important information todesign antitumor strategies involving IFN-a and anti-EGF-Rmonoclonal antibodies.

MATERIALS AND METHODS

Materials. DMEM and FBS were purchased from Flow Laboratories(Milan, Italy). Tissue culture plastic ware was from Becton Dickinson(Lincoln Park, NJ). Human recombinant interferon-«2b(2 x IO8units/mg) was a gift of Schering Corp. (Kenilworth, NJ). L-[35S]Methionineand [|:5I]EGF were obtained from Amersham Laboratories (Buckingh

amshire, England). Receptor grade EGF was purchased from Sigma(St. Louis, MO). Monoclonal antibody 108.1 for the EGF receptor wasa gift from Dr. Joseph Schlessinger, Rorer Biotechnology, Inc., Kingof Prussia, PA.

Cell Culture and Cell Proliferation Assay. The human epidermoidcarcinoma A43I cell line, provided by Professor F. Blasi (IstitutoInternazionale di Genetica e Biofìsica,Napoli, Italy), were maintainedin DMEM supplemented with 10% FBS, 20 mivi HEPES, penicillin-streptomycin, and L-glutamine. The human epidermoid carcinoma KBcell line, provided by Professor S. Bonatti (II Facoltà di Medicina,Universitàdi Napoli, Italy), was grown in DMEM supplemented withheat-inactivated 10% FBS, 20 mM HEPES, penicillin-streptomycin, L-glutamine, and sodium pyruvate. The cells were grown in a humidifiedatmosphere of 95% air/5% CO2 at 37°C.For cell growth experiments,A431 and KB cells were seeded at 2 x IO5 and 3 x IO5 cells/wellrespectively, in 6-well plates. After 24 h the medium was removed andfresh medium containing IFN-«was added then and every 48 h thereafter. At selected times cell growth assessment has been performed byhemocytometric cell count and trypan blue viability assay, after gentletrypsinization.

Flow Cytometric Analysis of Class I HLA Expression. Cells (1 x IO6)were seeded in 10-mm dishes and the treatment with IFN-« wasperformed for 48 h (see above). The cells were harvested with a

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a-IFN EFFECTS ON EGF-R

nonenzymatic solution [EDTA, 0.02% (w/v)], then washed twice withice-cold PBS, with the addition of 2% human serum. The MAb TEC-(32,an anti-ft-microglobulin, purchased from Techno Genetics (Milan,Italy), were added to the cells (10 M'/IO6 cells in 50-^1 volume), while

an irrelevant MAb has been used as negative control. After 30 min at4°Cthe cells were washed twice and incubated with 100 ß\of fluorescein

isothiocyanate goat-anti-mouse from Techno Genetics, diluted 1:20 at4°Cfor 30 min in the dark. The cells were then washed again twice and

resuspended in 500 /jl of PBS for flow cytometric analysis performedby a fluorescence-activated cell sorter (Becton Dickinson, Lincoln Park,NJ). Data analysis was performed by Consort 30 program, and lightscatter parameters (FSC-SSC) were used for live cell gating.

EGF Binding. A431 and KB cells were seeded in 24-multiwell platesat 2 x IO4and 5 x IO4cells/well, respectively. After 24 h, and every 48

h thereafter, growth medium was replaced with fresh medium containing IFN-a at the desired concentration. After overnight incubation inserum-free medium (DMEM supplemented with nonessential aminoacids and vitamins) in the presence or absence of IFN-«,the cells werewashed twice with ice-cold PBS supplemented with 1 mg/ml of BSA,and have been incubated for 3 h at 4°Cwith 200 ^I/well of binding

buffor (DMEM, 25 mM HEPES, 1 mg/ml BSA) containing 30,000cpm/well of ['"I]EGF (164 ^Ci/mg) and increasing concentrations ofunlabeled EGF, as previously described (23). After the 4°Cincubation,

the cells were washed 4 times with PBS/BSA and lysed in 0.5 ml/wellof 20 mM HEPES, 1% Triton X-100, and 10% glycerol. Cell-associatedradioactivity was counted in a Beckman gamma counter. The value ofEGF-R binding affinity and the receptor number were determined byScatchard analysis of the EGF-binding data (24), using EBDA/L1GAND, a computer program for fitting multiple binding site data(25).

Metabolic Labeling of EGF Receptor and Solubilization of IntactCells. Subconfluent A431 cells were grown in 100-mm dishes containing DMEM supplemented with 10% FBS in the presence or absence of500 units/ml IFN-«.The cells were starved for 30 min at 37°Cin

methionine-free medium with IFN-«.For radioisotope incorporationthe cells were incubated for 3 h in methionine-free medium with 200MCi/ml L-["S]methionine (1300 Ci/mM) with or without IFN-«.Cell

number was quantitated in unlabeled replicate dishes. For cell extractpreparation, the cells were washed twice with ice-cold PBS/BSA.scraped, and centrifuged for 30 min at 4°Cin 1 ml of lysis buffer ( 1%

Triton, 0.5% sodium deoxycholate, 0.1 M NaCl, l mM EDTA, pH 7.5,10 mM Na(PO4), pH 7.4, 10 mM phenylmethylsulfonyl fluoride, 25mM benzamidin, 1 mM leupeptin, 0.025 unit/ml aprotinin)/sample.The supernatants were subjected to immunoprecipitation with 108.1anti-EGF-R MAb. The EGF-R was precipitated from 200 M' of celllysate by using 10 ^\ of antibody (l h at 4°C)and 50 M'of 50% ProteinA-Sepharose suspension from Sigma (30 min at 4'C).

Electrophoresis and Autoradiography. Immunoprecipitated sampleswere washed 4 times with lysis buffer supplemented with 0.1% sodiumdodecyl sulfate, boiled for 5 min in Laemmli buffer (26) and electro-phoresed by 6.5% sodium dodecyl sulfate-polyacrylamide gel electro-phoresis. Molecular weight markers were purchased from Amersham.Th«gel was fixed for 30 min in 25% methanol-10% acetic acid,fluorographed for 30 min with Enhancer from NEN (Lachine, Quebec,Canada), dried on a Bio-Rad model gel drier, and finally exposed toFuji film at -80°Cfor autoradiography.

Immunoelectron Microscopic Analysis. Immunolabeling of EGF-Rswas performed on untreated or on IFN-«-treated cells (fixed in 2%formaldehyde at 4°Cin PBS for 2 h). The cells were incubated with0.1 -0.3 mg/ml of 108.1 MAb in PBS for 1 h at 4°C,washed extensively,and labeled for 3 h at 4°Cwith colloidal gold conjugated with Protein

A (Pharmacia Fine Chemicals, Uppsala, Sweden). Processing for electron microscopy and quantitative analysis was performed as previouslydescribed (27).

RESULTS

Effect of IFN-a on Growth and Class I HLA Expression ofA431 and KB Cells. Incubation of A431 and KB cells with IFN-a results in a marked inhibitory effect on cell growth; 48 h after

treatment with IFN-a, A431 cells show a higher percentage ofinhibition than KB cells (Fig. 1); the concentration inducing50% inhibition of cell proliferation is 500 units/ml for A431and 1000 units/ml for KB cells. The observed difference maybe due to the shorter doubling time of A431 cells as comparedto KB cells (24 h versus 48 h, respectively). The growth inhibitory effect is time and dose dependent. An increased growthinhibition is, in fact, observed at the 96-h time point (Fig. 1).It is noteworthy that growth inhibition occurs in the absence ofevident cytotoxicity, as assessed with trypan blue viability assayand without changes in cell cycle kinetics, as assessed by flowcytometric analysis (data not shown).

It has been widely reported that class I HLA are selectivelyenhanced by IFN-a in cultured tumor cells (19). Therefore weevaluated, by indirect immunofluorescence and flow cytometricanalysis, class I HLA expression, as a marker of specific IFNeffect, in untreated and in IFN-treated A431 cells. As expected,a 60% increase in the number of class I positive cells is observed48 h after the beginning of IFN treatment as assessed by indirectimmunofluorescence and flow cytometric analysis (data notshown).

IFN-a-induced Upregulation of EGF Binding on A431 and KBCells. We then examined the effect of IFN-a on EGF bindingto A431 and KB cells. To estimate EGF-binding capacity incontrol and in IFN-«-treated cells, increasing amounts of unlabeled EGF were allowed to compete for [I25I]EGF binding;

this method of analysis has shown an increase in the totalradioactivity specifically associated with IFN-a-treated cells.Consistent with these results, Scatchard analysis of the bindingdata shows about 2-fold increase in the receptor number of theIFN-a-treated cells, whereas no changes in the binding affinity

LUÜ

100

80

60

40

20

10 50 100a-IFN (U/ml)

500 1000

Fig. I. Effects of different concentrations of IFN-n on A431 (A) and KB (B)cell growth, evaluated 48 and 96 h from beginning of treatment. The cells wereseeded and grown overnight (see "Materials and Methods"); the day after, growth

medium was replaced and IFN was added from xlOO stock solutions then andevery 48 h thereafter. Growth inhibition is expressed as percentage of untreatedcontrol; points, mean of triplicate counts. SD never exceeded 5%. Cell viability,assessed by trypan blue, was always higher than 90%.

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0-IFN EFFECTS ON EGF-R

are detected (Fig. 2). Both cell lines express two classes ofsurface receptors with Kd values of 0.5 nM (high affinity) and4.7 nM (low affinity) for A431, and 0.08 nM and 2.5 nM for KBcells, respectively. Apparently only the number of low affinityreceptors is affected by IFN-a, while high affinity receptornumber remains unchanged (Table 1).

Time and Dose Dependency of IFN-a-induced EGF-R Up-regulation. IFN-a effect on EGF binding is time and dosedependent (Fig. 3). The observed effect is not an early event; itis detectable only after 24 h and occurs maximally 48 h afterbeginning of treatment with IFN-a (Fig. 3A). At later timepoints, EGF binding of IFN-treated cells approaches the controlvalues. A clear dose dependency results from dose-responseexperiments which were performed at the 48-h time point when

the maximal effect was observed (Fig. 3ß).Increased EGF-R Protein Synthesis in IFN-a-treated Cells.

As mentioned above, a possible mechanism for EGF-R upre-

3500

gulation could be the occurrence of increased receptor proteinsynthesis after IFN treatment of epidermoid cancer cells. Totest whether the synthesis of EGF-R is altered after exposureof A431 cells to IFN-a, we took advantage of the anti-EGF-R108.1 MAb which does not interfere with the EGF-bindingregion of the receptor. Twenty % confluent A431 cells weretreated with IFN-a for 48 h as described in "Materials andMethods," and then were metabolically labeled with [35S]me-

thionine for an additional 3 h. The labeled cell extracts werethen immunoprecipitated and the resulting samples were runon 6.5% polyacrylamide gel electrophoresis. It has been possibleto identify the immunoprecipitated receptor as a well-definedMr 170,000 band, which corresponds to the expected molecularweight of human EGF-R protein (1) (Fig. 4/4), Lanes 1, 2, 3,

and 4). The specific M, 170,000 band is due to a specificimmunoprecipitation as it is absent when unrelated controlMAbs are used.

2500 -

1500 -

UJO

Q.o

omu.om

500 -

eoo

400

200

0.01 0.1 10 100

UNLABELED EGF, nMFig. 2. Binding of ['"I]EGF to untreated (O) or IFN-a-treated (•)A431 (A) and KB (B) cells (500 units/ml and 1000 units/ml of IFN-a, respectively), in the

presence of different concentrations of unlabeled EGF. Points, averages from triplicate determinations. The insets show Scatchard plots of specific binding. Dataanalysis was carried out with a curve-fitting (multiple-site) Scatchard analysis computer program.

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a-IFN EFFECTS ON EGF-R

Table 1 Effect oflFN-a on EGF receptor number in A431 and KB cellsTreatment with 500 units/ml IFN-«was performed for 48 h. Receptor number

was estimated, utilizing a curve-fitting (multiple-site) Scatchard analysis computerprogram, from ['"I]EGF-bindingdata described in Fig. 2.

CelllineA431KBTreatmentNoneIFN-«

500units/mlNoneIFN-a

1000 units/mlHigh

affinityreceptors/cell4.1

XIO54.2X10'4.6

xIO44.8x IO4Low

affinityreceptors/cell3.2

x10"5.4x10'2.6

X10*3.7x 10'

250

200

150

100

—¿� 50O

oo'S

£a

Om

O

250 r

200 r-

150

100

50

O 6 12 24 48 72

TIME (hr)

B

96

O 10 100 500 1000a-IFN (U/ml)

Fig. 3. Time course (A) and dose response (B) experiments of IFN-«effectson EGF binding in A431 cells. ['"IjEGF specific binding was expressed as apercentage of that measured in control cultures. ['"I]EGF nonspecific binding

was determined in the presence of 100 nM unlabeled EGF. Points, average oftriplicate determinations.

We attempted thereafter a quantitation of the M, 170,000band by counting the radioactivity associated with the EGF-R-containing gel slices. Approximately a 2-fold increase in theEGF-R protein synthesis was detectable 48 h after the beginningof IFN treatment (Fig. 4B).

Increase in EGF-binding Sites Is Paralleled by IncreasedReceptor Protein Immunodetectable at Cell Surface. To testwhether the number of EGF-R molecules are actually increasedat the cell surface in IFN-a-treated cells, we performed electronmicroscopy immunodetection experiments with the anti-EGF-R 108.1 MAb.

Again, an increase of EGF-R protein is clearly observed atthe cell membrane of A431 IFN-a-treated cells compared tothe control (Fig. 5). This result argues for a true receptor proteinincrease, excluding receptor unmasking as a cause of upregu-lated expression. A quantitative assessment of the gold immu-nolabeling at the cell surface, performed by counting goldparticles per microns of cell plasma membranes, well agreeswith EGF-binding and immunoprecipitation data (Table 2). It

must be noticed that no major morphological changes resultedfrom A431 treatment with IFN-a.

In conclusion, the results from electron microscopy experiment indicate that the increase of EGF-R synthesis providesthe molecular basis for the observed upregulation of EGFbinding in the IFN-a growth-inhibited tumor cells.

DISCUSSION

We have shown that IFN-a induces evident growth inhibitionof A431 and KB human squamous carcinoma cells and enhances the expression of class I H LA, a specific marker of IFNaction. Radiobinding experiments have shown that the growthinhibitory response to IFN-a is accompanied by a time anddose dependent increase in the number of EGF receptors, whichis due to a specific increase in the receptor protein synthesis asassessed by immunoprecipitation. Moreover, the increasedEGF-R protein synthesis is accompanied by an increase inEGF-R antigens detectable on tumor cell surface. Notably,radiobinding, protein synthesis, and membrane receptor immunodetection experiments show quantitatively consistent results, suggesting a mechanistic link among all these events.

A large body of evidence shows the existence of various agentsable to modulate EGF-R expression. Among them, retinoicacid (12), Adriamycin (13), and IFN--y (14) have been described

200K-

96K-

68K-

45K-

234

B 20

16

O 12

1234Fig. 4. Effect of IFN-a on EGF-R protein synthesis. EGF-R was immunopre-

cipitated with 108.1 anti-EGF-R MAb from ["Sjmethionine-labeled A431 cells.A, 100 or 200 ¿ilof lysate from untreated cells (Lanes I and 2, respectively) and100 or 200 pi of lysate from cells which had been treated for 48 h with 500 units/ml IFN-a (Lanes 3 and 4, respectively) were immunoprecipitated with 10 n\ of108.1 MAb. On 6.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresisunder reducing conditions, the immunoprecipitated receptor appeared as a M,170,000 band. II. EGF-R band associated radioactivity was quantitated by counting the [3*S]EGF-R-containing gel slices. Histograms I, 2, 3, and 4 correspond toLanes 1, 2, 3, and 4, respectively. From replicate samples we also calculated theSDs, which were lower than 10% in both controls and IFN-n-treated cells.

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a-IFN EFFECTS ON EGF-R

Fig. 5. Effect of IFN-iv on the number ofEGF-Rs immunodelectable at A431 cell surface. Surface immunolabeling of prefixed cellswas performed with 108.1 anti-EGF-R M Ab.followed by the addition of Protein A colloidalgold conjugates: control (A); 500 units/ml IFN48-h-treated cells (A). (.-<)and (B), x 14.000;bar. I „¿�ni. ,-•¿�-'-.„¿�••

Xv *•*s'

K

Table 2 Quantitation of immunolabeled EGF-R on plasma membranes ofuntreated and IFN-a-treated A4ÃŒIcells"

urn Gold particles

ControlIFN-o 500 units/ml

61.3541.82

744950

12.01 (n = 8)23.18(n = 6)

°Data derived from immunolabeling experiment as described in Fig. 5 (TI=

number of micrographs counted).

to upregulate EGF-R expression in a number of systems. Onthe other hand, several agents like IFN-a (15) and retinoic acid(16) have been shown to inhibit EGF binding in other models.

Therefore, there is no clear picture of EGF receptor modulation, since different experimental models have been used and,so far, radiobinding data have not been correlated to proteinsynthesis or protein immunodetection studies. Interestingly,results from Zoon et al. (15) on bovine MDBK untransformed

cells, showed IFN-tv-induced EGF-R downregulation occurringbetween 8 and 20 h after beginning of treatment. However, acompletely different experimental model has been used in thisstudy as compared to our work, normal bovine versus humanepidermoidal cancer cells, respectively. It may be argued, therefore, a different effect of IFN on EGF-R expression in normalas compared to cancer cells, which often show an abnormallyhigh basal level of receptor expression (5, 6). This apparentdiscrepancy may suggest a novel and more selective way ofinterfering with tumor cell growth and therefore could be ofclinical interest.

Furthermore, the proliferative response to EGF does notappear to correlate with IFN-a effect on EGF-R expression, asMDBK and KB cells are growth stimulated by EGF, and A431are growth inhibited (15, 21, 22).

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rt-IFN EFFECTS ON EGF-R

At present it is difficult to understand the physiologicalmeaning of the IFN-a-induced upregulation of EGF-R as wellas the biological significance of a variety of other different IFN-ascribed effects (18, 20). The timing of the IFN-a-induced upregulation of EGF-R suggests that this effect could be secondaryto changes in the cell growth state, rather than being a directeffect of IFN-a on EGF-R synthesis. As a possible workinghypothesis, we therefore suggest that the increased receptorexpression could be part of a homeostatic cellular response toheavy antiproliferative stimuli. This hypothesis correlates withthe observation that growth inhibition induced by differentagents is accompanied, as described above, by EGF-R upregulation (12-14).

Several monoclonal antibodies, which have been raisedagainst EGF-R, are presently investigated as immunoreagentsfor tumor diagnosis and therapy (10, 28). Moreover, immuno-detectability with labeled anti-EGF-R MAbs of human cancercells, grown as tumors in the nude mouse, is proportional tothe basal expression of EGF-Rs (29). Therefore, upregulationof such receptors by IFN-a could be of practical interest,because it might result in an increased tumor targeting for drug-conjugated anti-EGF-R MAbs (30, 31).

Furthermore, concentrations of 500 or 1000 lU/ml of IFN-a, which are utilized in our in vitro experiments, can be easilyreached in humans (32), and eventually a more stable IFNconcentration can be obtained through locoregional administrations (33).

In this view, the understanding of both the basic mechanismand the timing of EGF-R upregulation are essential steps forthe design of clinical studies based on the use of IFN-a andanti-EGF-R antibodies. This requires a preliminary investigation about the effect of IFN-n on the cytotoxicity exherted invitro and in vivo by drug-conjugated anti-EGF-R MAbs on

human epidermoid cancer cells.

ACKNOWLEDGMENTS

We thank Drs. F. Ciardiello and G. Tortora for useful discussionand critical reading of the manuscript.

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1991;51:1294-1299. Cancer Res   Alfredo Budillon, Pierosandro Tagliaferri, Michele Caraglia, et al.  

-Interferon in Human Epidermoid Cancer CellsαUpregulation of Epidermal Growth Factor Receptor Induced by

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