(CANCER RESEARCH57. 3360-3364. August 15. 1997]

Advances in Brief

Two- and Three-dimensional Cell Structures Govern Epidermal Growth FactorSurvival Function in Human Bladder Carcinoma Cell Lines1

Virginie Dangles,2FrançoiseFéménia,VéroniqueLamé,Madeleine Berthelemy, Danielle Le Rhun,Marie-France Poupon, Daniel Levy, and Isabelle Schwartz-Cornil3

URA JNRA-DGER d'immunopathologie Cellulaire et Moléculaire.Ecole Nationale Vétdrinaire,94704 Maisons Alfort cedex (V. D.. F. F.. V. L, M. B., D. L R., D. L, I. S-C.], andinstitut Curie, 26 rue d'Ulm, 75005 Paris fM-F. P.], France

Abstract

Human bladder carcinomas often express high levels of the epidermalgrowth factor (EGF) receptor. In three human bladder carcinoma celllines (OBR, T24, and 647V), we show that two EGF receptor ligands,namely EGF and transforming growth factor a, enhanced the apoptosisdue to serum starvation on cells cultured as monolayers. Conversely, EGFand transforming growth factor a prevented apoptosis when the sameserum-starved cells were cultured as three-dimensional spheroids. Bothstimulation and inhibition of apoptosis by EGF were associated with p2!WAF1/CIP1 overexpression In 647V spheroids, EGF protection againstradiation-induced apoptosis was negated by genistein and tyrphostinAG1478, suggesting that blockade of the EGF signal transduction inpatients with bladder cancer may improve the radiotherapy efficacy.

Introduction

The mature tyrosine kinase EGFR4 is a Mr 170,000 transmembraneglycoprotein that is overexpressed in a number of human malignancies, including cancers of the lung, head and neck, brain, breast, andbladder (1). In human bladder carcinomas, EGFR overexpression hasbeen found in 58% of cases and has been associated with high tumorstages (2). Given that bladder tumor tissues are immersed in urine thatcontains two EGFR ligands, namely EGF and TGF-a (3, 4), it can beproposed that EGFR stimulation plays a role in bladder cancer tumorprogression.

The binding of EGF to its receptor induces the phosphorylation ofseveral tyrosine residues in its cytoplasmic tail (5). These phosphorylations serve as docking sites for proteins such as GAP, Grb2, andShc that couple the EGFR to the ras pathway, leading to the mitogenactivated protein kinase activation and the phosphorylation of specificnuclear transcription factors (5). This molecular pathway usuallyculminates in cell proliferation (5). However, the growth of some celllines such as the A431 line, which derives from a human epidermoidcarcinoma, is inhibited by EGF (5). The growth inhibition induced byEGF is mediated by STAT1 activation that up-regulates the cyclindependent kinase inhibitor p21 WAF1/CIP1 (6). Paradoxically, EGFstimulates the growth of A43 1 cells when they are cultured in multicellular three-dimensional spheroids or when they are implanted inmice, inferring that the culture conditions can modulate the responseto EGF (7). The overexpression of the EGFR has been considered toconfer a potent growth advantage to cancer cells; because cancer

Received 3/1 8/97; accepted 7/2/97.The costs of publication of this article were defrayed in part by the payment of page

charges. lists article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

I Supported by the Institut National de Ia Recherche Agronomique.2 Present address: Faculté des Sciences Pharmaceutiques et Biologiques, 4 avenue de

l'Observatoire, 75006 Paris, France.3 To whom requests for reprints should be addressed.

4 The abbreviations used are: EGFR, epidermal growth factor receptor; EGF, epider

mal growth factor; TUNEL, terminal deoxynucleotidyl transferase-mediated nick endlabeling; TGF-a, transforming growth factor a; FACS, fluorescence-activated cell sorting; GLM, general linear model.

arises from cells that survive and/or proliferate better than theirnormal counterparts, we assessed whether EGF and TGF-a modulatethe response to apoptotic signals in three bladder cancer cell lines(647V, OBR, and T24). We studied the effects of EGF and TGF-a onthe apoptotic response of serum-starved or irradiated bladder cancercells cultured as standard monolayers or three-dimensional spheroids,and we analyzed the associated modulation of p2! WAF1/CIP1 cxpression.

Materials and Methods

Cell Lines. The 647V, OBR, and T24 bladdercarcinomacell lines wereobtained from D. Chopin (Hôpital Mondor, Créteil,France), D. Bellet (Facultéde Pharmacie, Paris, France), and R. S. Kerbel (Sunnybrook Hospital, NorthYork, Canada), respectively. All of the cell lines were derived from humanepidermoid transitional bladder carcinomas. Immunocytochemical analyseswith an anti-EGFR antibody (clone E2760; Sigma, St. Louis, MO) establishedthat all cell lines express high levels of the EGFR (data not shown). Cells werecultured in RPMI 1640plus 10%FCS (Life Technologies, Inc., Paisley, UnitedKingdom) in a 5% CO2 atmosphere at 37°C.The cells were routinely foundnegative for mycoplasma infection (MycoTect; Life Technologies, Inc.).

EGF and TGF-a Treatment of Cells Grown as Monolayers. The bladder cancer cells were plated as 15 X i0@cells/cm2 in 6-well plates (Falcon,Lincoln Park, NJ) in 10% FCS for 16 h to allow cell attachment. The cells werethen washed twice in RPM! 1640 and further incubated for 48 h in RPM! 1640with and without human recombinant EGF (Life Technologies, Inc.) or human

recombinant TGF-ct (Sigma).EGF and TGF-a Treatment of Cells Grown as Spheroids. Dishes for

three-dimensional cell cultures were prepared as follows: 6 g of poly(2-hydroxyethyl metacrylate) were dissolved in 50 ml of 95% ethanol. This stockwas further diluted at 1:50 in 95% ethanol, and 1 ml was used to coat 6-wellculture dishes (Falcon). The dried wells were washed with PBS and furtherused for seeding 25 x l0@cells/mI of RPM! 1640 plus 10% FCS. After a 3-hincubation for the 647V cells and a 6-h incubation for the OBR and T24 cells,the cells formed spheroids that were harvested by centrifugation and werefurther incubated in serum-free RPM! 1640 plus or minus EGF or TGF-a for24—48h.

Radiation Experiments. EGF was added when the cells were plated inRPM! 1640 plus 10% FCS on the poly(2-hydroxyethyl metacrylate)-coateddishes to form spheroids. Genistein (Sigma), tyrphostin AG1478 (Sigma), orthe DMSO equivalent vehicle was added to the culture medium 3 h later; a10-gray irradiation (@°Cobomb) was performed 30 mm later. The treated andirradiated spheroids were then incubated for 24 h.

Apoptosis Detection and Cell Cycle Analyses For cell cycle analyses,adherent cells and spheroids were dissociated using trypsin digestion, washedin PBS, fixed in cold 70% ethanol, washed in PBS, treated with 50 pg/miRNase for 30 mm, incubated for 10 mm in 20 pg/mi propidium iodide(Sigma), and analyzed using a FACScan (Becton Dickinson) with exclusion ofcell doublets. The apoptotic nuclei appear as a subdiploid population. Alternatively, apoptosis was estimated using the TUNEL technique, performedaccording to the manufacturer's recommendation (Boehringer Mannheim,Mannheim, Germany).

Immunoblotting for p2! WAF1/CIP1 Detection Cells plated as monolayers for 16 h or as spheroids for 3 h in the presence of FCS were washed and

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EGF SURVIVALFUNCFIONIN HUMAN BLADDERCARCINOMA

ing a high level of the EGFR would show a high propensity to die inthe presence of EGF. We reasoned that this biological response toEGF was obtained in monolayer cell cultures, whereas the cancer cellsgrow in vivo as three-dimensional solid tumors. To mimic the tumorgeometry in vitro, cells were cultured as three-dimensional spheroids.The effect of EGF addition was tested on serum-starved 647V cultured in three-dimensional conditions. A high level of apoptosisdetected by TUNEL or by staining of the subdiploid nuclei wasobserved in the serum-starved spheroids (45%) compared to theserum-cultured spheroids (20.5%) after a 24-h culture (Fig. 2); theaddition of EGF or TGF-a potently prevented apoptosis and reducedit to 10% in both serum-starved and serum-cultured spheroids (Fig. 2).By 48 h, apoptosis in the 647V serum-starved spheroids increased to55%,whereasit wasmaintainedaslow as 12%in theEGF-treatedspheroids (Fig. 2B). When observed under light microscopy, theserum-free cultured spheroids appeared small with many sheddedcells, whereas the EGF- or the TGF-a-treated spheroids in the absenceof serum appeared as big bowls without shedded cells (Fig. 2E). Theseresults show that 647V spheroids are highly sensitive to serum deprivation when compared to 647V monolayers; in addition, the apoptosis in three-dimensional spheroids was potently prevented by EGFand TGF-a. EGF and TGF-a also supported a higher viability in theserum-cultured spheroids, thus allowing better anchorage-independentgrowth, a property associated with high malignancy (Fig. 2B).

The EGF addition to scram-starved spheroids was mainly associated with an accumulation of cells in the G@ phase from 37.5%without EGF to 67.5% with EGF at 24 h; EGF also led to a slightincrease in the percentage of cells going through the S-G2-M phasefrom 17.5% without EGF to 22.5% in the presence of EGF (Table 1).The cell accumulation in 0@ and the slight increase of cells in theS-G2-M phase were also observed in the spheroids treated with EGFin the presence of serum (Table 1).

When cultured as spheroids, the two other bladder cancer cell lines,OBR and T24, were similarly protected from cell death induced byserum deprivation, although to a lesser extent as compared to the647V cells (Fig. 2D).

further incubated in serum-free medium plus or minus 10 ng/ml EGF. Somecells were harvested before the cell wash to constitute the zero time point.

During serum starvation and EGF treatment, cells were harvested at severaltime points, and total cell extracts were prepared and denatured by boiling for5 mm in SDS-polyacrylamide sample buffer. Total cell lysate (50 @.tg)waselectrophoresed on SDS-PAGE and electrotransferred to Immobilon-P membranes (Millipore, Bedford, MA). The membranes were reacted with 1 pg/mianti-p21 WAF1/CIP1 (C-l9 clone; Santa Cruz Biotechnology, Santa Cruz,CA) and revealed with a 1:7500 dilution of a goat antirabbit IgG peroxidasecoupled antibody (Sigma). The protein products were visualized with enhancedchemiluminescence (Boehringer Mannheim). The densitometry analysis of the

signals was performed with the public domain NIH Image 1.60 software.

Results

EGF StimulatesApoptosisof Serum-starvedBladderCancerCells Cultured as Monolayers. Because EGFR is expressed at ahigh level in bladder cancer cells, we hypothesized that this may givea survival advantage to these cells, namely by conferring protectionagainst apoptosis. The bladder cancer cell line 647V was initially usedto test the effect of EGF or TGF-a on serum starvation-inducedapoptosis. Forty-eight-h serum-starved 647V cells were analyzed forthe detection of subdiploid nuclei and TUNEL-positive cells, and amuch higher level of apoptosis occurred in the EGF-treated cells(45%) as compared to the untreated cells (12%; Fig. lC). A cell cyclestudy on serum-starved 647V cells showed that EGF slightly reducedthe percentage of cells in the S-G2-M phase but drastically reduced thenumber of cells in G0-G1 from 46.5% in the control to 17.5% in theEGF-treated cells, suggesting that the cells died in G1 (Table 1). Noeffect on apoptosis or on the cell cycle was seen when the 647V cellswere cultured with 10% FCS (Fig. 1B; Table 1). Similar results wereobtained with TGF-a (Fig. 1A).

Two other bladder cancer cell lines, OBR and T24, were alsosensitive to the induction of apoptosis by EGF in serum-free conditions, albeit to a lower extent as compared to the 647V cells (Fig. 1D).

Three-dimensional Cell Structures Are Required for EGF Protection against Apoptosis Induced by Serum Starvation. Thus far,the results suggest the conclusion that aggressive cancer cells express

A

C

Fig. 1. EGF enhances apoptosis in serumstarved 647 bladder cancer cells cultured as monolayers. A, serum-starved 647V cells were treatedfor 48 h with increasing amounts of EGF (0) orTGF-a (•);the apoptotic nuclei were detected bypropidium iodide staining. Serum-cultured (B) andserum-starved (B and C) 647V cells were treatedwith 10 ng/ml EGF for 48 h, and apoptosis wasdetected by propidium iodide staining (B) andTUNEL detection (C). D, serum-starved 647V,OBR, and T24 monolayer cultures were treated for48 h with 10 ng/ml EGF and assessed for apoptosisdetection using propidium iodide staining. The resuits (A, B, C, and D) are the means and SDs fromduplicated wells. The GLM procedure of the SASsoftware showed that the differences between control versus EGF-treated cells were statistically different (significant level, <0.05) except for theOBR cell line (D) that displayed the same overalltendency.

-I-

0 IÔ 20@ 3Ô 40EGF (0)orTGFct(•),ng/mI + +

- + - +

Cl) 30Cl)

120

@210

EGF

42%

0 . @J@@@2:r OBREGF- + -+ - +TUNEL (FL-i)

3361

60

50(I)

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10

f@t4

B40

.@ 30

0

0.

FCSEGF

D @o

II@ controlE

TUNEL (FL-i)

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P!

Table 1 Effects of the EGF treatment on the647V cell cycle in two- or diree-dimensionalculturesTwo-dimensional

culturS'Three-dimensionalcultures'Serum

No serum

Control EGF Control EGFSerum Control EGFNo

serum

Control EGF10

graysc

ControlEGFCellcycleaphaseSub-G1

(%)G0-G1 (%)S-G2-M (%)8.5

±0.7 8.1 ±0.1 12.0 ±1.4 45.0 ±2.851.0 ±1.0 50.5 ±0.5 46.5 ±0.7 17.5 ±2.140.0 ±2.0 41.0 ±1.4 40.5 ±0.7 37.5 ±0.720.5

±2.1 10.0 ±1.451.0 ±0.5 59.0 ± 1.4

28.5 ±0.7 31.0 ±0.045.0

±2.8 10.0 ± 1.437.5 ±2.1 67.5 ±2.1

17.5 ±0.7 22.5 ±0.745.0

±5.0 17.0 ±3.522.5 ±3.5 38.0 ± 1.4

33.0 ± 1.4 45.0 ±2.1

EGF SURVIVAL FUNCTION IN HUMAN BLADDER CARCINOMA

A5o B40

j30

@ I01620364050

EGF( o)orTGFa(•),ng/mI

C D

FCS ++ - - - -EGF -+ - + -+

Fig. 2. EGF prevents apoptosis in serum-starved or irradiated bladder cancer cells cultured as spheroids. A, serumstarved spheroids were treated for 24 h with EGF (0) orTGF-a (•),and apoptosis was detected by propidium iodidestaining. B, serum-cultured and serum-starved spheroids werecultured with and without 10 ng/ml EGF for 24 or 48 h andprocessed for apoptosis detection using propidium iodidestaining. C, serum-starved spheroids were treated for 24 hwith 10 ng/ml EGF and processed for TUNEL reaction andFACS analysis. D, serum-starved 647V, OBR, and T24 spheroids were treated for 24 h with 10 ng/ml EGF and analyzedfor apoptosis using propidium iodide staining of the apoptoticnuclei. E, light microscopy examination (X 10) of serumstarved 647V cultured spheroids with (2) and without (1)EGF. F. 647V cells were plated as spheroids in 10% FCS for3 h in the absence or presence of EGF and then irradiated with10 grays. Genistein, tyrphostin AG1478, or DMSO was applied to the spheroids 30 mm before irradiation. Apoptosisdetection was performed by propidium iodide staining of thepermeabilized nuclei. The results (A. B, C, D. and F) are themeans and SDs from duplicated wells. The GLM procedureof the SAS software revealed that the EGF and genisteineffects were statistically significant (significant level, <0.05)except those obtained with the T24 cell line (D) that showedthe same tendency.

control EGF

.10%

- 647V OBR T24

EGF - + - + - +

F50

40U)

@20@

E1

2

0_,‘yrays + + + + +_+ +

EGF(ng/m@) 10 20 20 20 20 20Genlstein (@tg/mI) - - 30 - - -

AG1478(xlOOnM) 1.5 3 5

EGF Up-Regulates p21 WAF1/CIP! Protein Expression Bothin Monolayerand in Three-dimensional647V Cell Cultures.Because p21 WAF1/CIP1 has been shown to be involved in the promotion of some apoptotic responses (8) and in EGF-mediated growthinhibition (6), we investigated whether its expression was modulatedby EGF in the 647V cells cultured as monolayers. Compared to 647Vcells cultured in serum, the p21 WAF1/CIP1 level of expression inserum-starved 647V cells showed a 3-fold down-regulation at 4 h toan 8-fold down-regulation at 48 h, whereas the EGF-treated serumstarved 647V cells showed a sustained p21 WAF1/CIP1 level (Fig. 3).

It can thus be concluded that EGF up-regulates the p21 WAF1/CIP1level of expression when 647V cells are serum-starved in monolayerconditions.

The expression of p21 WAF1/CIP1 regulated by EGF differedwhen the 647V cells were cultured as three-dimensional spheroids.After a 3-h culture in serum, the spheroids were serum-starved andtreated with EGF for several periods of time. We observed that a 4-hserum starvation was accompanied by a 3-fold decrease in p21WAF1/CIP1 that persisted for 24 h, whereas EGF maintained p21 WAF1/CIP1 expression at a high level that was even enhanced after 18 h.

a Cell cycle analyses were performed on 24-h (spheroid) and 48-h (monolayer) cultures using propidium iodide staining of the permeabilized nuclei. FACS analyses using Lysis

II software gave the percentage of cells in sub-G1 (apoptosis), in G0-G1 and S-G2-M phases. The results are means and SDs from two independent experiments. The GLM procedureof the SAS software (18) revealed that the EGF effects were statistically significant (significant level, <0.05).

b 647V cells were cultured in the presence or absence of serum in monolayers (two-dimensional culture) or spheroids (three-dimensional culture) and treated with or not treated with

10 ng/ml EGF.C Serum-cultured 647V spheroids were incubated with EGF (20 ng/ml) for 3 h before irradiation (10 grays).

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Time 0 4h 24h 48h In the presentstudy,we investigatedwhetherEGFor TGF-awould modulate the response of the human 647V bladder cancercells to apoptosis. We found that the 647V bladder cancer cellculture geometry determined the apoptotic response to serum starvation, with three-dimensional spheroids being more sensitive thanmonolayers. This difference reveals that the cellular dependenceon survival factors present in serum is modulated by the tissue

O.D.X1@ 12 4 7 4.7 15 6 20 culturearchitecture.WethenobservedthatTGF-aIEGFpromotedserum starvation-induced apoptosis when the cells were cultured asmonolayers, whereas TGF-aIEGF strongly prevented apoptosis inthree-dimensional spheroids. These opposite effects of EGF onapoptosis induction were also observed, although to a lesser extent,with two other bladder cancer cell lines, T24 and OBR. Suchopposite effects of EGF have been reported for the A43 1 humansquamous cell carcinoma that is growth-inhibited by EGF in monolayer cultures and stimulated when established as spheroids (7).EGF infusion into animals bearing A43 1 tumors stimulated ratherthan inhibited growth (7). This latter observation, together with thenotion that spheroids better reproduce the tumor microenvironmentthan do two-dimensional cultures (7, 9, 12), strongly supports thatcell cultures in spheroids allow the evaluation of tumor response togrowth factors. Molecular investigations aimed at delineating thebasis of the alternative response to EGF focused on the EGFR levelof expression and on its autophosphorylation (7). Growth inhibition in response to EGF has been correlated to overexpression ofthe EGFR in several cell lines; the EGFR expression in A43 1 cellsis 20-fold higher in monolayers as compared to spheroids (7). Thekinetics and the overall level of the EGFR autophosphorylationdiffered in monolayer cultures versus spheroids, implying differences in the resulting signal transduction. We also found that 647Vbladder cancer cells presented lower levels of the EGFR in spheroids as compared to monolayers by using immunostaining andFACS analyses (data not shown). However, this difference did notpotently alter the EGFR Signal transduction pathway leading to p21WAF1/CIP1 expression; in both culture conditions, serum starvation resulted in down-regulation of p21 WAF1/CIP1, whereas EGFtreatment maintained its expression at a high level. This finding isin agreement with previous reports that showed an up-regulation ofp21 WAFI/CIP1 by EGF in the DiFi human colon carcinoma,MDA468 human breast cancer, and ME18O and A43l humancervical squamous carcinoma cells in a mechanism that seemsindependent of p53 (6, 13); however, the up-regulation of p21WAF1/CIP1 in A43l cells was associated with inhibition of cellgrowth, whereas in 647V cells, the up-regulation of p21 WAF1/CIPI by EGF was encountered in both promotion or prevention ofapoptosis, indicating that p21 WAF1/CIPI alone does not determine death or survival.

Several reports mentioned conflicting results regarding p21 WAF1/CIP1 and apoptosis; for instance, the knockout of the p21 WAFJ/CIPJgene in the HCT1 16 colorectal cancer cell line resulted in promotionof apoptosis after Adriamycin treatment (14); on the contrary, p21WAF1/CIP1 overexpression in glioma GB-I cells increased theirsusceptibility to cisplatinum (8). The mechanisms underlying theopposite effects of p21 WAF1/CIP1 are unclear but suggest that p21WAF1/CIP1 can be a key molecule in the apoptotic response, depend

A blockedtheEGF-mediatedprotectionagainstradiation-inducedapoptosis (Fig. 2F). This result strongly suggests that the inhibition ofapoptosis by EGF is a consequence of the EGFR tyrosine kinase

O.D.X1@ 14 15 4.5 15 3.2 15 2.1 activationandthatinhibitorsoftheEGFRcouldbeusedtosensitize- . cancer cells to radiation.

p21WAF1/CIP1@ @5@ • #@DiscussionEGF - + •+ •+

p21WAF1/CIP10@@@@@EGF-- +- +- +

Time 0 4h 18h 24h

Fig. 3. A, 647V cells cultured as monolayers in 10% FCS for 24 h (time 0) wereserum-starved and incubated with (+) or without (—)10 ng/ml EGF for several periodsof time. B, 647V cells were cultured as spheroids for 3 h in 10% FCS (time 0) and thenincubated with (+) or without (—)10 ng/ml EGF for several periods of time. Total proteinlysates were subjected to Western blotting analysis using an anti-p21 WAF1/CIP1antibody revealed by chemiluminescence. The scanned signals were quantitated usingNIH Imagei.6 software, and the estimated absorbances are reported. This experiment wasrepeated three times, all of which gave similar results.

Overall, p21 WAF1/CIP1 expression was differentially regulated byserum starvation and EGF in spheroids versus monolayers; serumstarvation decreased p21 WAF1/CIP1 expression more potently in themonolayers, and EGF increased the p21 WAF1/CIP1 level morestrongly in spheroids. In conclusion, the up-regulation of p21 WAF1/CIP1 by EGF in serum-starved 647V cells is unusual because it doesnot lead to cell cycle arrest but is rather associated with the inductionof apoptosis in two-dimensional cultures and protection against apoptosis in three-dimensional cultures.

EGFProtects647VCellsAgainstRadiation-inducedApoptosisin Three-dimensionalSpheroids,and EGFRTyrosineKinaseInhibitors Abrogate Its Effect. Three-dimensional conditions are oftenused to assay the radiation sensitivity of cells (9). Because radiationtherapy is routinely used in bladder cancer treatment, we assessedwhether EGF would have adverse effects in protecting against radialion-induced apoptosis in 647V cell spheroids. A 10-gray irradiationon 647V preformed spheroids in serum-containing medium induced45% apoptosis and a cell accumulation in G2-M phase, a commonfinding in bladder cancer cells (Ref. 10; Fig. 2F; Table 1). The EGFtreatment (20 ng/ml) initiated before irradiation led to a significantprotection against apoptosis (down to 17%) with cell accumulation inboth G1 and G2 (Fig. 2F; Table 1). Thus, it seems that the protectiveeffect of EGF is not linked to a specific phase of the cell cycle. Thisresult suggests that the overexpression of the EGFR in bladder cancercells associated with the presence of a high level of EGF and TGF-ain the bladder tissue microenvironment may impair the efficiency ofradiation therapy.

Tyrosine kinase inhibitors (1 1) have been used to block signaltransduction from tyrosine kinase receptors. When 647V cells growing as spheroids in the presence of EGF were preincubated withgenistein at 30 ,.Lg/ml before irradiation, the protection against apoptosis by EGF was abrogated, and a high level (47%) of irradiationinduced apoptosis was obtained (Fig. 2F). The highly selective EGFRinhibitor tyrphostin AG1478 used at 150—500nM also efficiently

EGF SURVIVALFUNCtiON IN HUMAN BLADDERCARCINOMA

B

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EGF SURVIVALFUNCTIONIN HUMANBLADDERCARCINOMA

ing on the cell context. In that respect, an increase of the p21WAF1/CIP1 level induced apoptosis in mammary epithelial cells thatwere actively cycling and did not affect arrested cells, illustrating theemerging concept that conflicting growth signals lead to apoptosis(15). In our bladder cancer model, we found a marked difference inthe cycling activity between serum-starved 647V cells cultured asmonolayers and spheroids, with over 40% of cycling cells in themonolayers and around 17% in the spheroids. We propose that EGFinduced p21 WAF1/CIP1 overexpression may result in either apoptosis or protection against apoptosis, depending on the cell cycleactivity associated with the culture geometry of the bladder cancercells.

The doses of EGF and TGF-a that we used are close to thephysiological range of the concentrations found in vivo, with around2 and 10 ng/mg of bladder cancer tissue of EGF and TGF-a, respeclively (3), indicating that the effects we observed may apply to the invivo situation.

We also report in this study the inhibition of EGF protectionagainst radiation-induced apoptosis by two EGFR tyrosine kinaseinhibitors, genistein and tyrphostin AG1478. Genistein inhibits theEGFR autophosphorylation when used at a 10—50 @g/mlconcentration and has been shown to delay or prevent experimentalcancers in animals (16); however, genistein is not very specific ofthe EGFR tyrosine kinase activity and may affect other signaltransduction pathways (1 1). We showed a total reversal of theEGF-mediated protection by using the highly specific EGFR inhibitor tyrphostin AG1478 (1 1, 13), indicating that the specificinhibition of the EGFR tyrosine kinase activity is enough tosensitize cells to radiation. Although the experiments were donewith the 647V cell line only, our results encourage us to proposethe use of EGFR inhibitors as an adjuvant treatment to radiotherapy against cancers, namely bladder cancers that express highlevels of the EGFR. Interestingly, a previous work by Baselga eta!. (17) indicated that combination therapy with EGFR antagonistantibodies and doxorubicin can actually have a potent antitumoreffect on xenografts. These antitumor effects were attributed to theadditive growth suppression of doxorubicin and anti-EGFR antibodies; the impact on apoptosis was not investigated (17).

Altogether, our data show that the apoptotic response of serumstarved bladder cancer cells to EGF is differentially regulated by thetissue culture architecture, despite a constant increase of p21 WAF1/CIPI expression. Because spheroids reproduce an important part ofthe tumor environment, it is likely that the protective effect of EGFencountered in multicellular three-dimensional cultures reflects theEGF response in tumors. The overexpression of the EGFR encountered in bladder cancer cells may confer a survival advantage tothe tumor cells and thus contribute to the aggressiveness of tumorbehavior.

Acknowledgments

We are especially indebted to P. Devauchelle and J. P. Cotard for theirstrong support and to the scanner team for easy access to the @°Cobomb

usually used to treat canine and feline cancers at the Ecole Nationale Vétérinaire d'Alfort. We warmly thank D. Bellet for being so helpful and for the giftof the OBR cell line. We acknowledge R. S. Kerbel for inspiring this study and

for the gift of the T24 cell line. We thank D. Chopin for the 647V cell line andP. Lescoatand M. Saanafor statisticalanalyses.

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1997;57:3360-3364. Cancer Res   Virginie Dangles, Françoise Féménia, Véronique Lainé, et al.   Cell LinesGrowth Factor Survival Function in Human Bladder Carcinoma Two- and Three-dimensional Cell Structures Govern Epidermal

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