VoL 7, 173-178, February 1996 Cell Growth & Differentiation 173

Epidermal Growth Factor-induced Apoptosis in A431 Cells CanBe Reversed by Reducing the Tyrosine Kinase Activity1

Laith F. Gulli, Kenneth C. Palmer, Yong Q. Chen,and Kaladhar B. ReddY�

Department of Pathology, Wayne State University, Detroit, Michigan

48201

Abstract

A431 cells overexpress epidermal growth factorreceptors (EGF-Rs) and are inhibited by EGF. We showthat treatment of A431 cells with 10 n� EGF induced a15-fold increase in EGF-R autophosphorylation, leadingto inhibition of cell proliferation and morphologicalfeatures of apoptosis. However, at a lowerconcentration of EGF (0.01 nM), there is a 2-foldincrease in EGF-R autophosphorylation and increasedcell proliferation when compared to untreated cells.EGF treatment is associated with increased expressionof c-myc and decreased expression of mutant p53 andp21/WAF protein. When A431 cells were simultaneously

treated with 10 n� EGF and EGF-R antibody, there wasa significant reduction in EGF-R autophosphorylationthat was associated with increased cell proliferation.Based on these results, we postulate thatoverexpression of EGF-R could allow for selectivegrowth advantage for tumor cells in the presence ofnormal or decreased ligand availability. However,excessive ligand binding would result in deregulatedgrowth signaling, leading to growth inhibition andprogrammed cell death.

IntroductionThe mature EGF-R is an Mr 1 70,000 transmembrane glyco-protein that is overexpressed in a number of human malig-nancies including cancers of the lung, head and neck, brain,bladder, and breast (1). EGF3 and TGF-a are natural ligandsfor EGF-R that initiate cellular responses by binding to spe-cific receptors on the surface of target cells (2, 3). Likeseveral other growth factors, the binding of EGF to its cellsurface receptor triggers a cascade of intracellular events,

including induction of a tyrosine kinase activity intrinsic to the

EGF-R. Shortly after binding, the EGF-R complexes are lo-

Received 7/1 1/95; accepted 11/17/95.The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to mdi-cate this fact.1 This work was supported by a grant from the National Cancer Institute(to K. B. A.) and the Pathology Department, Wayne State University.2 To whom requests for reprints should be addressed, at Department ofPathology, Wayne State University, 540 E. Canfield, Detroit, Ml 48201.Phone: (313) 577-6191 ; Fax: (313) 577-0057.3 The abbreviations used are: EGF, epidermal growth factor; EGF-R, EGFreceptor; TGF, transforming growth factor.

calized in clathrin-coated regions of the plasma membraneand are then internalized by the cell (4). These events lead toincreased DNA synthesis and cell proliferation in most of thecells. A431 cells have high levels of EGF-R, but mitogenicresponse to EGF does not correlate with their increased EGFbinding capacity. Thus, while low concentrations of EGFstimulate cell proliferation, high (1 0 nM) EGF concentrations,which are mitogenic for many other cell lines, inhibit cellproliferation in A431 cells (5, 6). Because EGF stimulatesgrowth of several types of cell in culture (4), it has beenproposed that activation of tyrosine kinase mediates theproliferative effect. It has also been shown that EGF inhibitsproliferation of GH4C1 rat pituitary tumor cells (7), A431 hu-man epidermoid carcinoma cells (5, 6), and certain humanbreast cancer cells (8, 9). This indicates that the relationshipbetween biological effects of EGF and tyrosine kinase activ-ity is complex.

Apoptosis, or programmed cell death, is an active processrequiring protein synthesis and specific endonucleolytic di-

gestion of cellular DNA (10). Cellular proliferation depends onthe ability of cells to respond to external mitogenic stimuli,

including hormones and growth factors. This response ismediated through a cascade of signal transduction eventsultimately leading to an alteration in nuclear gene expression

and cell proliferation (11-15). There is compelling evidencethat membrane and cytoplasmic enzymes involved in proteinphosphorylation, initially recognized for their roles in cellcycle progression, are also central to the transduction ofapoptotic signals. Two recent studies contradict the involve-ment of tyrosine kinase in the apoptotic process. Pre-B cells

that are transformed by a temperature-sensitive allele of thev-abl oncogene arrest in G1 and rapidly undergo apoptosis

when the abl-encoded tyrosine kinase is inactivated bygrowth at the nonpermissive temperature (1 6). In contrast,enhanced tyrosine phosphorylation of multiple substrates inhuman B-lymphocyte precursors has been observed upon

induction of apoptosis in response to ionizing radiation (17).

In the present study, we examined the effects of activated

EGF-R tyrosine kinase on cell proliferation and apoptosis inA431 cells. We show that excessive ligand binding wouldincrease tyrosine kinase activity, and this results in deregu-lated growth signaling, leading to programmed cell death.We went on to show that by partially inhibiting the tyrosinekinase activity either by EGF-R antibodies or by tyrosinekinase inhibitor RG-1 3022, we were able to partially reversethe cell death.

ResultsEGF-R Antibody Blocks EGF-induced Cell Inhibition inA431 Cells. We have examined the effect of blockade of theEGF-R that mediates the effects of EGF. To block EGF-R, we

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Fig. 1. Effect of EGF-R antibody on on EGF-stimulated autophosphory-lation of the EGF-R in A431 cells. A431 cells were grown in DMEM, with10% serum, to near-confluence. The seeding medium was removed; thencells were washed twice with PBS, and the medium was replaced withphenol red-free and serum-free DMEM with 0.1 % BSA. After 24 h, cellswere stimulated with EGF in the presence or absence of EGF-R antibody(monoclonal antibody 225) at a concentration of 1 5 hg/mI. EGF wasadded for 3 mm, and EGF receptor phosphorylation was evaluated byWestern blot analysis, as described in “Materials and Methods.”

174 Regulation of Apoptosis by Tyrosine Kinase Activity

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used an EGF-R antibody (monoclonal antibody 225), whichblocks the EGF binding domain of the EGF-R (1 8). Similar toprevious reports using other cell lines, EGF-R antibody in-hibited the phosphorylation of the EGF-R in A431 cells (Fig.

1). Having demonstrated that EGF-R antibody can block

receptor autophosphorylation, we asked whether antibody

could rescue the EGF-induced growth inhibition in A431cells. �H1Thymidine incorporation and cell number were an-alyzed in the presence of high concentration of EGF with orwithout EGF-R antibody (1 5 �g/ml). In the presence of anti-body, there was a significant reduction in receptor autophos-phorylation and increased thymidine incorporation and cellproliferation when compared to EGF-alone treatment cells(Fig. 2). This observation supports the hypothesis that theEGF-induced growth inhibition in A431 cells is due to theincreased tyrosine kinase activity, and these effects can bepartially reversed by reducing the tyrosine kinase activity.

Effect of EGF-R Phosphorylation on A431 Cell Prolifer-ation and Viability. Treatment of A431 cells with 1 0 nt�i EGF

showed a 15-fold increase in EGF-R phosphorylation, andthis resulted in sustained inhibition of cellular proliferation,

followed by a decline in viable cell number after 18 h oftreatment. We noted that >80% of the treated cells contin-

ued to exclude trypan blue at 24 h, indicating that the earlyeffect of EGF on these cells is not due to loss of membraneintegrity. If the toxicity of EGF is mediated via increasedtyrosine kinase, then such toxicity should be prevented by asimultaneous exposure to EGF and tyrosine kinase inhibi-tors. To test this hypothesis, experiments were performedusing 1 0 nM EGF and 60 n� RG-1 3022 tyrosine kinase inhib-itor. It was shown previously that tyrphostins (RG-1 3022) arepotent inhibitors of tyrosine kinase activity of EGF-R and cellproliferation in a variety of target cells in vitro (15, 1 9-21). Toconfirm that these compounds can block EGF-R autophos-

phorylation in A431 cells, we examined the effects of RG-

1 3022 on A431 cells that contain relatively large numbers of

EGF-R. After a 3-mm exposure of these cells to EGF, West-em analysis using antiphosphotyrosine antibodies demon-strated a significant inhibition of EGF-stimulated autophos-

phorylation of the Mr 1 70,000 band representing the EGF-R(Fig.3).

We next investigated the effects of reduced tyrosine ki-nase activity on cell viability in A431 cells after 3 days inculture. As seen in Fig. 4, the growth curve for the cellsexposed simultaneously to 10 nM EGF and 60 n� RG-1 3022show a >30% increase in the cell viability when compared toEGF treatment alone. These data show that by inhibiting theEGF-induced tyrosine kinase, cell viability can be signifi-cantly increased.

Evidence of Apoptosis in EGF-treated A431 Cells. Cells

exposed to 10 nM EGF exhibited cytoarchitectural features ofapoptosis, as determined by fluorescence microscopy, afterstaining with ApopTag. Untreated cells and those treatedwith a low concentration of EGF (0.01 nM) exhibited normalmorphology, with spontaneous expression of apoptotic traits

seen in <2% of the cells scored (Fig. 5). In contrast, mani-festation of prominent apoptotic morphology, i.e., cell shrink-age, condensation of nucleoplasm and cytoplasm, and for-mation of membrane blebs and membranous apoptoticbodies was evident by 8 h. By 1 6 h, >1 5% of the cells

showed apoptosis, and by 3 days, over 80% of the cells died.In the presence of EGF-R antibody, the number of cellsundergoing apoptosis was reduced to 6-7% by 16 h. Theseresults demonstrate that the growth-inhibitory/cytotoxic ef-fects of EGF on A431 cells are associated with changes in

cell morphology, consistent with apoptosis. We were unable

to demonstrate DNA ladder formation associated with ap-optosis in A431 cells. It was shown previously that A431 cellsexposed to UV-B irradiation have shown DNA fragmentation

(22). Because we were unable to show DNA ladder formationin EGF-treated A431 cells, we repeated the ApopTag three tofour times. We consistently produced the same results.

Effect of EGF on c-myc, p53, and p21/WAF Expressionin A431 Cells. Since increased expression of early responsegenes has been intimately associated with key functions inthe control of cell growth, proliferation, and programmed celldeath in several models (23-30), we examined A431 cells forthe effect of high and low EGF on the immediate-early proto-oncogene c-myc. Fig. 6 demonstrates that high EGF treat-ment is associated with a 10-fold induction of c-myc. Thec-myc induction was seen after 15 mm of EGF treatment andcontinued to increase, even after 2 h. At a lower concentra-tion of EGF treatment, there is 2-3-fold induction of c-myc.

The p53 (mutant) and p21/WAF protein was accumulated upto 4 h, and by 24 h, the amount of p53 and p21/WAF had

decreased to the level of untreated cells (Fig. 7). Theseresults suggest that growth inhibition associated with EGF isindependent of p53/p21/WAF protein. High c-myc inductionby EGF (10 nM) with simultaneous growth inhibition is asso-ciated with apoptosis in A431 cells.

Discussion

The present study demonstrates that A431 cancer cells,which possess an unusually large number of EGF-Rs per cell,are growth inhibited by EGF and undergo programmed celldeath over a period of time. The characteristic loss of cellularproliferative capacity, morphological changes, such as cellshrinkage, condensation of nucleoplasm and cytoplasm, and

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Cell Growth & Differentiation 175

Fig. 2. Effect of EGF and monoclo-nal antibody 225 (AB 225) on DNAsynthesis in A431 cells. Cells grow-ing in exponential phase in phenolred-free/serum-free medium werestimulated with different concentra-tions of EGF in the presence or ab-sence of monoclonal antibody 225 ata concentration of 15 �g/ml. Rate ofDNA synthesis was estimated at18 h. values are the means of tripli-cate determinations; bars, SE.

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Fig. 3. Effect of RG-13022 on EGF-stimulated autophosphorylation ofthe EGF receptor. Cells were grown as shown in Fig. 1 . After 24 h,RG-13022 or DMSO (Control) was added to the cultures. After an addi-tional 1 h, EGF was added for 3 mm, and EGF receptor phosphorylationwas evaluated by Western blot analysis, as described in “Materials andMethods.”

formation of membrane blebs and membranous apoptotic

bodies are seen following exposure of these cells to 1 0 nMEGF, while low concentrations of EGF (0.01 nM) stimulate

their proliferation. This correlation suggests that the mere

presence of a large number of EGF-Rs has no effect on

apoptosis, but that increased tyrosine kinase activity medi-

ates programmed cell death in this cell population. If thishypothesis is correct, phosphorylation of tyrosine residues in

proteins may result in either stimulation of cell proliferation or

programmed cell death. A reduction in EGF-stimulated ty-

rosine kinase activity appears essential for the escape from

growth inhibition and subsequent apoptosis.

Precise analysis of the quantitative relationship between

EGF-stimulated tyrosine kinase activity and cell growth is

difficult. A reduction in EGF-R autophosphorylation was

achieved by tyrosine kinase inhibitor RG-1 3022 (15, 1 9) or by

monoclonal antibody against EGF-R. The reduction in ty-

rosine kinase activity may be due to EGF-R antibodies corn-

petitively inhibiting EGF binding and/or down-regulating

EGF-R by internalization, without stimulating receptor phos-phorylation (18). A 30-40% reduction in tyrosine kinase ac-

tivity appears to be sufficient, not only for escape from

growth-inhibitory effects, but also by reducing the number of

cells that undergo apoptosis. It is possible that an apoptotic

pathway is activated in the cells because receptors and

tyrosine kinase activity are excessively elevated, leading to

deregulated growth signal. When EGF-stimulated tyrosine

kinase is reduced, activation of the inhibitory pathway may

no longer predominate.

The cellular proto-oncogene c-myc and wild-type p53

were shown to be involved in cell proliferation and pro-

grarnmed cell death in fibroblast LM-3 cells (29). An elegant

study of activation-induced death of T cells, using antisense

oligonucleotides to c-myc, demonstrated that c-myc expres-

sion is a necessary component of the apoptotic process

seen with T-cell receptor activation (26). In the same study,

however, the authors reported that c-myc antisense had no

effect on dexamethasone-induced apoptosis in the same cell

line, suggesting that glucocorticoid-induced and activation-

induced apoptosis may proceed through different pathways.

It was shown previously that A431 cells have a mutant p53

(31 , 32). These facts bring into question the role of c-myc

expression and mutant p53 in apoptosis of A431 cells. Our

11

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176 Regulation of Apoptosis by Tyrosmne Kinase Activity

breast cancer cell lines since increased tyrosine kinase

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Fig. 4. Effect of RG-13022 on EGF-stimulated cell inhibition and viability.A431 cells were plated in DMEM with serum. Twenty-four h later, theseeding medium was removed; the cells were washed twice with PBS,and the medium was then changed to phenol red-free and serum-freeDMEM, with or without inhibitor. The cells were counted on days 1 , 2, and3, and the percentage of viable cells was determined by trypan dye-exclusion assay. 0, control; 0, 0.01 n� EGF; V, RG-13022; O , 10 nM EGF;., 10 nM EGF+ RG-13022. The figure shows the results of mean ± SE oftriplicate determinations. All SEs were <5%.

results with high and low EGF show a distinct pattern ofc-myc induction. The observation that cells exhibiting a mi-

togenic response to low EGF and those exhibiting an apop-totic response to high EGF have a similar pattern of imme-

diate-early gene induction suggests that these genes initiate

a signal whose specificity is regulated by some downstreamdeterminants, depending on the intensity of the signal. A

second possibility is that c-myc can act as a bivalent regu-lator, determining either cell proliferation or apoptosis, de-pending on whether free movement around the cell cycle issupported by growth factor or limited by growth factor de-privation or treatment with other cell cycle-blocking agents(33). Our studies suggest that high EGF concentrations in-hibit A431 cell proliferation with simultaneous induction of

c-myC and leads to apoptosis that is independent of p53(mutant) and p21/WAF. In contrast, at low EGF concentra-

tions, cell proliferation was not inhibited with increased

c-myc induction, allowing cell cycle reentry and cell prolifer-

ation instead of apoptosis.

In conclusion, our results lend support to the idea that

A431 cells overexpressing EGF-R are inhibited by high

levels of EGF and eventually undergo apoptosis while

maintaining a proliferative response to low EGF levels (5,

9, 30). These findings support an analogous hypothesis in

can drive two coupled functions, proliferation and pro-grammed cell death. Based on the data reported here, wepostulate that excessive ligand binding would increasetyrosine kinase levels, and this results in deregulated

growth signaling, leading to growth inhibition and pro-grammed cell death. We went on to show that by loweringtyrosine kinase levels either by tyrosine kinase inhibitor orby blocking EGF binding to the EGF-R by EGF-R antibod-ies, we were able to reduced the tyrosine kinase activityand also partially interfere with apoptotic pathways. Highlevels of EGF-R in malignant cancers might give a growth

advantage to the cells due to extremely low levels of EGF,in serum, ranging from 20 to 27 �LM (34), and in tissue,ranging from 1 to 5 ng/g tissue (35). Based on our data, wesuggest that blocking the binding of EGF to EGF-R notonly interrupts the proliferative response but may also

interfere with apoptotic pathways.

Materials and MethodsCells and Cell Culture. The A431 cell line was obtained from the Amer-ican Type Culture Collection (Rockville, MD). A431 cells were cultured in

DMEM (GIBCO Laboratories, Grand Island, NY) supplemented with 10%FCS (GIBCO). All cell lines were routinely tested for Mycoplasma contam-ination and were not infected.

Growth Factors, Antibodies, and Tyrosine Kinase Inhibitor. EGFand TGF-a were purchased from Collaborative Research Laboratories(Lexington, MA). The tyrosmne kinase inhibitor, RG-13022, was purchasedfrom Biomol (Plymouth Meeting, PA). RG-13022 stock solutions weremade in DMSO and diluted to appropriate concentrations in culture me-

dium prior to the addition to the cells. An equivalent dilution of DMSO(0.1 %) without the inhibitor served as a control. EGF-R antibody andantiphosphotyrosine antibody were purchased from UBI (Lake Placid,NV). p53 antibody was purchased from NeoMarkers (Fremont, CA). p21/WAF was obtained from PharMingen (San Diego, CA).

Immunoprecipitatlon. A431 cells were plated in 175 flasks and, when70-80% confluent, were incubated in serum-free medium overnight.

Monolayers were then treated with EGF or TGF-a for 3 mm. Cells were

then washed with ice-cold PBS and scraped into lysis buffer [50 m�iTris-HCI (pH 7.6), 100 mM NaCI, 2 mr�i EDTA, 1% NP4O, 1 m�i orthovana-date, 0.5% sodium deoxycholate, 10 mg/mI leupeptmn, 2 mt�i phenylmeth-ylsulfonyl fluoride, and 10 mg/mI aprotinin]. After removal of cell debris by

centrifugation (1 4,000 x g for 30 mm), protein was estimated. Equalamounts of protein from different groups were subjected to EGF-R im-munoprecipitation or immediately boiled for 5 mm in sample buffer. Im-munoprecipitates were washed three times with HNTG [20 m�i Tns-HCI(pH 7.6), 10% glycerol, 0.1% NP4O, and 150 m�i NaCI] and heated inloading buffer for 10 mm (100#{176}C).Proteins were resolved by 8% SDS-

PAGE for EGF-R and p53 and 16% for p21/WAF, transferred onto nitro-

cellulose, probed with the appropriate antibody, washed three times withTIBS [20 mM Tris-HCI (pH 7.6), 0.05% Tween 20, and 100 mp�i NaCl], andincubated with a secondary antibody coupled to horseradish peroxidase.

After three washes, the filter was developed using the ECL chemilumi-nescence system (Amersham Corp.). The level of phosphorylated EGF-Rwas determined by densitometnc scanning of the M� 170,00 band.

Northern Hybridization. Total RNA for the Northern analyses wasprepared as described previously (36, 37). ANA concentration was deter-mined by measuring absorbance at 260 nm/280 nm. The concentrationand integrity of RNA preparations were confirmed by electrophoresis informaldehyde gels with ethidium bromide staining. ANA was electropho-

resed into a formaldehyde-i .2% agarose gel and blotted onto nitrocellu-lose (38). The nitrocellulose filters were hybridized with a 32P-labeled

cDNA in Rapid-hyb buffer, according to the manufacturer’s protocol (Am-ersham, Arlington Heights, IL). Relative levels of specific mRNA werequantified by densitometry.

DNA Synthesis. Cells were plated in 24-well tissue culture dishes

(Corning) at a density of 1 x i0� cells/well in DMEM with 10% FCS. After24 h, the seeding medium was removed; then the cells were washed two

Cell Growth & Differentiation 177

Fig. 5. Effect of EGF treatment onmorphology of A43i cells. Cell mor-phology as determined by phasecontrast microscope. Control, un-treated cells (A) and cells treatedwith 1 0 nM EGF/ml for 24 h (B). Cellmorphology as determined by im-munofluorescence microscopy af-ter staining with FITC-conjugatedanti-digoxigenin antibody of con-trols (C) and cells treated with 1 0 nMEGF (D). Photomicrographs showpositive fluorescence in cells thathave incorporated digoxigenin-la-beled dUTP by 3-OH end exten-sion of nucleosome fragments, mdi-cating apoptotic change.

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Fig. 7. Level of p53 and p21 /WAF protein after EGF stimulation in A431cells. Cells growing in exponential phase in phenol red-free/serum-freemedium were stimulated with EGF. p53 and p2i/WAF protein were eval-uated at different time points by Western blot analysis.

Fig. 6. c-myc mRNA expression in A431 cells following treatment with0.01 nM and 10 nM EGF. After treatment with EGF for 0, 15, 30, 60, or 120mm, total RNA (1 5 pg/lane) was isolated, size fractionated, and blottedonto nylon membrane. The blots were hybridized with 32P-labeled cDNAprobes, and autoradiographs were developed.

times with PBS and incubated in 0.1 % BSA and phenol red-free DMEM(1 mI/well). Twenty-four h later, EGF or AG-i 3022 and/or EGF-A antibodywere added. After 16 h, 0.25 �Ci [�H]thymidine (82.3 Ci/mmol; NENproducts, Boston, MA) in a volume of 25 Ml was added to each well for a1-h pulse. Cells were harvested, and the rate of DNA synthesis was

estimated in triplicate by measuring the acid-precipitable radioactivity asdescribed previously (37). Radioactivity was quantified in a Beckmann LS

7000 liquid scintillation counter with an efficiency of 64%.Monolayer Growth Assay. A43i cancer cells were plated in 6-well

tissue culture plates (Coming Laboratories, Houston, TX) at a density of 1x io� cells/well in DMEM containing 10% FCS. After 24 h, the seedingmedium was removed; then cells were washed two times with PBS, andthe medium was replaced by phenol red-free DMEM medium supple-mented with 0.1 % BSA. The medium was exchanged with fresh mediumcontaining the same supplements on the third day. The cells were counted

in triplicate on a hemocytometer after suspending the cells with 1 m�EDTA in PBS on day 5.

Analysis of Apoptotic Cell Morphology. For morphological exami-nation, cells were grown on coverslips. Subsequent to fixation, cells were

stained to identify apoptotic cells, according to the protocol provided withthe ApopTag kit (Oncor, Gaithersburg, MD).

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