[CANCER RESEARCH 55. 1883-1893, May 1, 1995]

Autocrine Transforming Growth Factor a Is Dispensible for v-rasHa-induced

Epidermal Neoplasia: Potential Involvement of AlternateEpidermal Growth Factor Receptor Ligands1

Andrzej A. Dlugosz,2 Christina Cheng, Erin K. Williams, Nadine Darwiche, Peter J. Dempsey, Bruce Mann,

Ashley R. Dunn, Robert J. Coffey, Jr., and Stuart H. Yuspa

Laboratory of Cellular Carcinogenesis and Tumor Promotion, Division of Cancer Etiology, National Cancer Institute, Bethesda, Maryland 20892 [A. A. D., C. C„E. K. W.,N. D., S. H. YJ; Departments of Medicine and Cell Biology, Vanderbill University School of Medicine, Nashville, Tennessee 37232 ¡P.J. D., R. J. C.¡;and Ludwig Institute forCancer Research, Royal Melbourne Hospital, Victoria 3050, Australia [B. M., A. R. DJ

ABSTRACT

Autocrine epidermal growth factor receptor activation by transforminggrowth factor a (TGFa) has been implicated in growth stimulation duringepithelial neoplasia. Using keratinocytes isolated from mice with geneticdefects in TGFa expression, we tested whether TGFa is required fortransformation by the v-ras"" oncogene. Introduction of v-ras"" into

primary epidermal cultures using a retroviral vector stimulated growth ofboth control (TGFa +/+, BALB/c) and TGFa-deficient (TGFa -/-,ira-I i keratinocytes. Moreover, v-ra.v"1' elicited characteristic changes in

marker expression (keratin 1 was suppressed; keratin 8 was induced),previously shown to be associated with epidermal growth factor (EGF)receptor activation, in both TGFa +/+ and TGFa -/- keratinocytes.v-ra.v"" markedly increased secreted o 10-fold) and cell-associated (2-3-

fold) TGFa levels in keratinocytes from TGFa +/+ and BALB/c mice,but not TGFa —/—or wa-l mice. Based on Northern blot analysis, \-ras""

induced striking up-regulation of transcripts encoding the additional EGFfamily members amphiregulin, heparin-binding EGF-like growth factor,

and betacellulin in cultured keratinocytes from all four mouse strains.Interestingly, in addition to the normal 4.5-kilobase TGFa transcript,wa-l keratinocytes expressed two additional TGFa transcripts, 4.7 and 5.2kilobases long. All three transcripts were up-regulated in response to\-ras"", as well as exogenous TGFa or keratinocyte growth factor treat

ment, and were also detected in RNA isolated from wa-l brain and skin.In vivo, v-rasH" keratinocytes from control as well as TGFa-deficient mice

produced squamous tumors when grafted onto nude mice, and theselesions expressed high levels of amphiregulin, heparin-binding EGF-like

growth factor, and betacellulin mRNA, regardless of their TGFa status.These findings indicate that TGFa is not essential for epidermal neoplasiainduced by the v-ra.v"1'oncogene and suggest that another EGF family

member(s) may contribute to autocrine growth stimulation of ra.v-trans-

formed keratinocytes.

INTRODUCTION

The majority of malignancies affecting humans arise in the epithelial component of organs including the skin, breast, lung, and gastrointestinal and genitourinary tracts. Both clinical data and results ofstudies using animal models indicate that epithelial neoplasia is astepwise process driven by the accumulation of multiple genetic andepigenetic changes (reviewed in Ref. l). Chemical carcinogenesis inmouse skin has provided a powerful experimental model for studyingboth early changes involved in the development of premalignantlesions, as well as later events important in neoplastic progression (2).Application of a subtumorigenic dose of the chemical carcinogen

Received 12/1/94; accepted 2/24/95.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 in accordance wilh18 U.S.C. Section 1734 solely to indicate this fact.

1This work was supported in part by a Thomas B. Fitzpatrick-Kao Corporation

Research Award (A. A. D.), and by Grant MCICA46413 and by the Veterans Administration (R. J. C.). R. J. C. is a Veterans Administration Clinical Investigator.

2 To whom requests for reprints should be addressed, at LCCTP/NCI, Bldg 37/Rm

3B08, 37 Convent DR MSC 4255, Bethesda, MD 20892-4255.

DMBA3 to mouse skin results in mutation of the c-ra.vHa gene (3).

Repeated treatment with the phorbol ester 12-O-tetradecanoylphor-bol-13-acetate promotes the clonal expansion of cells expressing the

mutant ras gene to form a benign tumor capable of autonomousgrowth. The central role of ras in the pathogenesis of benign papil-

lomas was corroborated in studies where introduction of oncogenicras into wounded skin could replace chemical initiation by DMBA(4), as well as nude mouse experiments in which primary keratinocytes transduced with v-rasHa in vitro produced tumors when grafted

as a reconstituted skin (5). In addition to its involvement in earlystages of cutaneous neoplasia, raíhas also been implicated in malignant progression of skin tumors (reviewed in Ref. l). Since mutationof cellular raígenes has been detected in a variety of human cancers(6, 7), understanding how activated Ras influences keratinocytebiology is likely to provide insights relevant to human disease.

Numerous studies have documented alterations in growth factorsignaling pathways during epithelial neoplasia, suggesting that thesechanges may be important in the development and/or maintenance ofthe neoplastic phenotype. TGFa and its receptor, EGFR, appear toplay a particularly prominent role in epithelial neoplasia (reviewed inRefs. 8 and 9). Expression of v-raiHa in cultured mouse keratinocytes

induces a large increase in TGFa mRNA and protein expression (10,11). In addition, high levels of TGFa have been detected in papillo-mas produced either by grafting v-raiHa keratinocytes onto nude mice

(10), or generated using a two-stage chemical carcinogenesis protocol(12-14). Treatment of normal keratinocytes in vitro with exogenousTGFa elicits many of the same phenotypic alterations as v-raíHa(11),

suggesting that oncogenic raímay alter keratinocyte behavior, at leastin part, via activation of the TGFa/EGFR signaling pathway. Consistent with this concept, transgenic mice in which TGFa is targetedto the epidermis using keratin 14 (K14) or keratin 1 (Kl) promotersdevelop squamous papillomas following tumor promotion by wounding or 12-O-tetradecanoylphorbol-13-acetate treatment (15, 16), suggesting that overexpression of TGFa can substitute for raíHagene

activation as an initiator in two-stage cutaneous carcinogenesis. It hasalso been suggested that TGFa can function as a tumor-promotingstimulus, based on the results of two-stage chemical carcinogenesis

studies (13) and grafting experiments using papilloma cell lines in anenvironment where TGFa is overexpressed (17). The development ofcutaneous papillomas and carcinomas following DMBA treatment ofa TGFa transgenic mouse strain (18) supports the concept that TGFacan function as a tumor promoter. In addition to TGFa and EGF,several other structurally related proteins bind and activate the EGFR,including amphiregulin (19), HB-EGF (20), and betacellulin (21). Therole of these "alternate" EGFR ligands in the physiology of normal or

neoplastic keratinocytes has not yet been defined.

3 The abbreviations used are: DMBA, 7,12-dimethylbenz[a]anthracene; EGFR, epi

dermal growth factor receptor; TGFa, transforming growth factor a; HB-EGF, heparin-binding epidermal growth factor-like growth factor; KGF, keratinocyte growth factor;CM, conditioned medium; TBS, Tris-buffered saline (20 mM Tris, pH 7.4-500 IHMNaCI);poly(A)+ RNA, polyadenylated RNA; PL-7, ribosomal protein L7.

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EOFR LÕGANOS AND EPIDERMAL NEOPLASIA

To test whether TGFa expression is obligatory in ras-mediatedepidermal neoplasia, we have studied the effects of v-rasHa on kera-

tinocytes from two mouse strains deficient in TGFa expression, thewa-1 spontaneous mutant (22) and the TGFa-null mouse, generated

by homologous recombination (23). Our findings argue against anobligatory role for autocrine TGFa in epidermal neoplasia and raisethe possibility that another EGFR ligand(s) may be capable of substituting for TGFa to stimulate the growth of v-rasHa-transformed

keratinocytes.

MATERIALS AND METHODS

Reagents. Recombinant human TGFa, KGF, and PCS were purchasedfrom UBI (Lake Placid, NY).

Mouse Strains and Cell Culture. Primary epidermal keratinocytes wereobtained from newborn mouse skin following overnight flotation on trypsin, asdescribed previously (24). In some experiments, keratinocytes were isolatedfrom adult mouse skin using a slightly modified protocol (25). Cells wereobtained from the following mouse strains: ABP/Le (The Jackson Laboratory,Bar Harbor, ME), a linkage strain homozygous for the wa-l mutation, which

arose spontaneously in a mouse colony of unknown genetic background (22);BALB/c (or BALB/cByJ,The Jackson Laboratory), a representative "normal"

strain; TGFa —/-, homozygous TGFa-null mice produced by homologous

recombination (23); and TGFa +/+, the TGFa-null control strain. TGFa +/+and TGFa —/—mice were generated on a C57BL6 background (23). In

addition to the wa-\ alíele,ABP/Le mice are homozygous for the followingrecessive mutations: a, non-agouti; b, brown; bt, belted; p, pink eye; and se,

short ear.Medium was prepared with Eagle's MEM (without Ca2+ and Mg2+) sup

plemented with 8% PCS [Ca2+-depleted (24)] and 0.25% penicillin-streptomycin solution (GIBCO, Grand Island, NY). The concentration of Ca2* in the

medium was adjusted by addition of 0.3 M CaCl2 stock. Cells were cultured inmedium with 0.05 mM Ca2 * to maintain a proliferating population of basal

cells. Primary keratinocytes were transduced with a defective retrovirus harboring the v-ra.cHa oncogene as described previously (5). Growth factor treat

ments were initiated 3 days after plating and continued until cells wereharvested for RNA isolation, which was performed 8-10 days after plating.For preparation of keratinocyte CM, cells grown in 60-mm culture dishes were

washed with PBS and 2 ml of fresh medium containing 1% PCS were addedto dishes. Medium was removed after approximately 24 h, centrifuged at1000 x g to remove any floating cells and culture debris, and stored at —20°C

until RIA. For the analysis of cell-associated TGFa, cultures were washed with

PBS, and the attached cells remaining in the dish were frozen on dry ice andstored at -20°C until processed for RIA. Where indicated, keratinocytes wereinduced to terminally differentiate by raising the Ca2+ concentration from 0.05

to 0.12 mM for 48 h (26).Immortalized cell lines were established from v-ra£H"-transduced TGFa

+/+ and TGFa —/—keratinocytes as follows. Primary \-rasHa keratinocyteswere trypsinized, pelleted, resuspended in medium containing 1.4 mM Ca2+

medium, and plated into 6-well culture dishes that had been precoated with asolution of fibronectin-collagen (25). As an alternative approach, floating cellsfrom v-ra.v"a-transduced primary cultures were collected, resuspended in medium with 1.4 mM Ca2+, and plated in precoated 6-well dishes. One to 2 daysafter plating, cells were washed with Car* and Mg2+-free PBS and replenishedwith medium containing 0.05 mM Ca24 medium. Keratinocytes were propagated continuously in 0.05 mM Ca2 * medium. A similar protocol was used toestablish an immortalized cell line, designated wa-lrp, from v-rasH"-trans-

duced H'a-1 primary keratinocytes. The SP-1 cell line was established from

SENCAR mouse papillomas (27). The C57r cell line was generated usingprimary v-ra.cHa keratinocytes obtained from newborn C57BL mice, and was

generously provided by Dr. Wendy Weinberg (Laboratory of Cellular Carci-

nogenesis and Tumor Promotion/National Cancer Institute).Growth Curves. Cells were plated in 6-well tissue culture plates (Costar,

Cambridge, MA) at a density of 3 X 10s cells/well in 2 ml of medium. The

following day, cultures were washed with PBS and fresh medium was added.Additional medium changes were performed on alternate days, and cells weretransduced on day 5 in culture using a retrovirus containing \-ras"* (5). To

assess keratinocyte growth, cells from replicate wells were trypsinized on

alternate days and counted using a Coulter Counter (model ZB1; CoulterElectronics, Hialeah, FL).

Skin Grafting. Tumorigenicity was tested by grafting a slurry of keratinocytes (2.5-6 x IO6 cells) and SENCAR mouse fibroblasts (5-8 X IO6 cells)

onto prepared graft sites on the backs of nude mice, as described previously (5,28). This procedure results in the development of a benign squamous papil-loma at the graft site when v-ra.?Ha-transduced keratinocytes are tested,

whereas normal skin is produced when control keratinocytes are grafted (5). Atthe completion of experiments, animals were sacrificed and tumors wereprocessed for routine histológica! analysis. For in situ hybridization, tissue wasalso embedded in OCT (Miles, Elkhart, IN), frozen by flotation in an ethanol/dry ice bath, and stored at -70°C.

Tissue for RNA isolation was used either fresh or frozen in liquid nitrogenand stored at —70°Cuntil processing (see below).

Radioimmunoassay and Antibodies. A RIA specific for murine TGFa(29) was used to measure immunoreactive TGFa in CM from mouse keratinocyte cultures and cell lysates. A polyclonal sheep antibody was raised to ratTGFa (East Acres Biologicals, Southbridge, MA) which was also used asstandard and 125I-labeled tracer. Dose-response curves of immunoreactiveTGFa detected in CM from normal and v-rasHa-transformed mouse keratino

cyte cultures were parallel to rat TGFa standards in the TGFa RIA. Levels ofmouse TGFa are therefore expressed in equivalent units of rat TGFa standard.For determining the level of cell-associated TGFa, cells in 60-mm dishes were

harvested in 1 ml of buffer containing 25 mM Tris (pH 8.0), 50 mM NaCl, 0.5%NP40, 0.5% sodium deoxycholate, 5 pig/ml leupeptin, 5 fig/ml aprotinin, and2 mM phenylmethylsulfonyl fluoride. Lysates were vortexed, extracted on icefor 45 min, vortexed again, and centrifuged at 16,000 X g for 10 min.Supernatants were used for determination of cell-associated TGFa by RIA.

The generation and specificity of rabbit anti-peptide antibodies recognizing

keratins Kl and K14 were described in a previous report (30). For immunoblotanalysis, anti-Kl was used at a dilution of 1:1000 and anti-K14 was diluted1:15,000. A rat monoclonal antibody, TROMA-1 (31), was used at a dilution

of 1:500 to detect keratin K8. This antibody was a gift from Dr. Rolf Kemler(Max Planck Institute, Freiburg, Germany), generously provided by Dr. IvanDamjanov (Thomas Jefferson University, Philadelphia, PA).

Polyacrylamide Gel Electrophoresis and Immunoblotting. Cultures werewashed with PBS and whole cell extracts were prepared by harvesting cells in lysisbuffer [0.25 M Tris (pH 6.8), 5% SDS, and 20% 0-mercaptoethanol] and heatingthe lysates at 100°Cfor 5 min. Proteins were separated in 8.5% polyacrylamide

gels for determination of keratin expression and electrophoretically transferred toreinforced nitrocellulose (BA-S 83) (Schleicher & Schuell, Keene, NH) in a buffer

containing 25 mM Tris (pH 8.3), 192 mM glycine, and 20% methanol for l h at 100V. Nitrocellulose blots were stained with Ponceau S (Sigma, St. Louis, MO) todetermine uniformity of protein loading and transfer (32). Following blocking for30 min with 5% milk in TBS, blots were exposed to primary antibodies for l h atroom temperature. Filters were washed for 30 min using three changes of TBSwith 0.2% Tween 20 (Bio-Rad Laboratories, Richmond, CA) and incubated withhorseradish peroxidase-conjugated secondary antibody, diluted 1:5000 in 2% milk

in TBS, for 30 min. Filters were washed as before, and immunoreactive proteinsdetected using enhanced chemiluminescence reagents purchased from Amersham(Arlington Heights, IL).

RNA Isolation and Northern Blot Analysis. Poly(A)+ RNA was isolated

from cells growing in 150-mm tissue culture dishes as described previously

(33). Total RNA from epidermal tumors or normal mouse tissues was preparedby homogenization in a guanidine isothiocyanate-phenol solution (TRIzol;

GIBCO, Gaithersburg, MD) based on a previously published protocol (34). Insome cases, total RNA served as a source for poIy(A)+ RNA isolation using

a modification of a protocol described earlier (33). RNA species were separated by electrophoresis through a 1% agarose gel containing 0.66 M formaldehyde and 0.66 fig/ml ethidium bromide and transferred to reinforced nitrocellulose (BA-S NC; Schleicher and Schuell, Keene, NH) (35). Transcript

lengths were determined using rRNA as markers. The following probes wereused, labeled with 32P by nick-translation, for Northern blot analysis: rat

TGFa, full-length cDNA (36); glyceraldehyde phosphate dehydrogenase, full-length cDNA (37); keratin 1 (Kl), 400-base pair cDNA (26); 400-base pair ratamphiregulin, 1.5-kilobase rat HB-EGF, 650-base pair mouse betacellulin, and784-base pair mouse EOF cDNA fragments (38). A 380-base pair cDNAfragment recognizing mRNA encoding PL-7 (39) was labeled by random

priming.

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EGFR LÕGANOS AND EPIDERMAL NEOPLASIA

BALB/c TGFCC+/+ TGFa-/-

Control

M-ras Ha

B107

LU

EI 1°6CO

HiO

105

-I- control

1 3 5 7 9 11 13 15 17

DAYSFig. 1. The v-rasHa oncogene induces similar morphological changes and hyperproliferation in normal and TGFa-deficienI primary epidermal keratinocytes. In A, cultured

keratinocytes obtained from normal (BALB/c, TGFa +/+) and TGFa-deficient (wa-1, TGFa —/—)newborn mouse skin were transduced with a defective retrovirus containing thev-rasH" gene as described in "Materials and Methods." v-rasH" keratinocytes from all mouse strains exhibited increased cell density and an extended culture life span, when comparedto controls. X 80. B, growth of TGFa +/+ and TGFa —/—keratinocytes under basal culture conditions and following transduction by the v-rasHa oncogene. Primary keratinocyteswere grown in 6-well tissue culture plates (plating density, 3 X IO5 cells/well) as described in "Materials and Methods." Cell counts were performed following trypsinizalion on the

indicated days. Points, mean values based on counts from three wells; bars, SEM.

In Situ Hybridization. In situ analysis was performed using 35S-labeled

neo riboprobe as described previously (40), based on a previously publishedprotocol (41).

RESULTS

TGFa Is Dispensible for Phenotypic Alterations and IncreasedCell Growth Induced by the v-r<wHaOncogene in Vitro. To assess

whether TGFa is required for phenotypic alterations induced by

oncogenic ras, control and TGFa-deficient primary epidermalkeratinocytes were transduced with a defective retrovirus containing v-ras"" (v-rasHd keratinocytes) as described in "Materials andMethods." BALB/c mice were used as a representative "control"

strain for the wa-l mutant, since the genetic background of thisstrain is unknown (22), while TGFa +/+ and TGFa -/- mice

have the same C57BL genetic background (23). In primary epidermal cultures, v-rasHil produced similar changes in cell morphology regardless of mouse strain. Keratinocytes in \-rasH" cultures

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EOFR LÕGANOS AND EPIDERMAL NEOPLASIA

Control v-rasHa

r I I

l.05 .12 .05 .12

I I I.05 .12 .05 .12

K1

K8

K14

Fig. 2. v-raiHa induces similar changes in keratin expression in TGFa null (-/-) andcontrol ( + /+) keratinocytes. Primary control and v-ra.vHakeratinocytes from the indicatedmouse strains were grown in medium with 0.12 mM Ca2+ for 48 h to induce terminal

differentiation. Western blot analysis was performed using total lysates as described in"Materials and Methods." v-ra.cHa induced similar alterations in keratin expression in both

normal and TGFa-null keratinocytes. The constitutively expressed keratin K14 was usedas a control for loading.

were generally more crowded than in corresponding controlcultures and exhibited a more phase-dense cytoplasm with prominent, phase-lucent intercellular spaces (Fig. IA). The growth ofcontrol and v-rasHa-transduced keratinocytes was assessed by per

forming cell counts over a 2-week period. From day 7 to 15 inculture, the number of cells in control TGFa —/—cultures was

significantly lower than in control TGFa +/+ cultures (Fig. 15),suggesting that endogenous TGFa plays a role in maintainingkeratinocyte growth under basal conditions in vitro. Transduction

with v-rasHa stimulated growth of keratinocytes of both genotypes;

interestingly, despite their slower growth under control conditions,TGFa —/—v-rasHa keratinocytes achieved the same final celldensity as TGFa +/+ v-rasH" keratinocytes (Fig. IB).

We have previously reported that v-rasHa keratinocytes induced to

terminally differentiate fail to express the spinous cell keratins Kl andK10 but aberrantly express keratins K8 and K18, and these changes inkeratin expression appear to be mediated by autocrine stimulation ofthe EGFR (11). Immunoblot analysis presented in Fig. 2 indicates thatv-rasHa induces comparable alterations in the keratin expression pat

tern in both TGFa +/+ and TGFa —/—keratinocytes. In controlkeratinocytes, raising extracellular Ca2+ from 0.05 to 0.12 mM in

duces expression of the differentiation-specific keratin Kl; in contrast,v-rasHa-transduced keratinocytes of either strain fail to express Kl but

synthesize K8 instead (Fig. 2). K14, which is constitutively expressedin cultured keratinocytes, is expressed at similar levels in all treatmentgroups.

TGFa Is Not Detected in Normal or v-ras"a-transformed Cul

tured Keratinocytes from TGFa-null Mice and Is Markedly Reduced in v-rasHa-transformed wa-1 Keratinocytes. Since v-rasHa

keratinocytes from all mouse strains examined exhibited phenotypicchanges previously attributed to activation of the EGFR signalingpathway, the levels of secreted and cell-associated TGFa were determined in control (BALB/c, TGFa +/+) and TGFa-deficient (wa-1,TGFa -/-) cultures (Fig. 3). Secreted TGFa in CM from either

BALB/c or wa-l control cultures was below the level of detection,

which was 39 pg/ml for this assay (Fig. 3A). TGFa secretion waselevated to 490 ±20 (SEM) pg/ml in CM from v-rasHa-transformed

BALB/c keratinocytes; in contrast, the level of TGFa in CM fromwa-l v-rasHa keratinocyte cultures was marginally increased, to87 ±2.5 pg/ml (Fig. 3A). v-ra.vHa also induced dramatic changes in

the level of cell-associated TGFa in BALB/c, but not wa-l, kerati-

B525-

£ "50J0 375-

9t300

1 8 225K 150-

j BALB/c

ras contr

TREATMENT

^2zmTGFa */+••TGFa •/-

ras contr

TREATMENT

ras contr

TREATMENT

contr

TREATMENT

Fig. 3. Induction of secreted and cell-associated TGFa by the v-rasH" oncogene is blocked in wa-l and TGFa null keratinocytes. A, wa-l keratinocytes. The level of TGFa wasdetermined by RIA in CM (2 ml/60-mm dish) collected following a 24-h exposure to control or v-rasHa-transduced primary keratinocytes. Cell-associated TGFa was measured in cellextracts prepared from keratinocytes attached to the substratum, as described in "Materials and Methods." Results are presented as mean values based on determinations performedusing three dishes; bars, SEM; B, TGFa-null primary keratinocytes; C, immortalized v-raiHa-transformed keratinocyte cell lines. Keratinocyte cell lines were established followingv-ru.!Ha transduction of epidermal cultures isolated from TGFa -/- mice (lines a-1, a-2, and a-6) or the parental mouse strain TGFa +/+ (line a + 2), as described in "Materialsand Methods." TGFa levels were assayed in CM and cell lysates for each cell line as described above. , RIA "background," determined using medium not previously exposed to

cells (for CM) or lysis buffer (for cell lysates).

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EOFR LÕGANOS AND EPIDERMAL NEOPLASIA

BALB/c iva-1

TGFa

GAPDH

u.o O CD <O I- s¿ >

HB-EGF

GAPDH ...*

GAPDH

Fig. 4. Expression of transcripts encoding multiple EGFR ligands is coordinatelyregulated in cultured BALB/c and wa-l keratinocytes. Primary cultures were either grownunder basal conditions (Control), transduced with the v-r«.vll;loncogene, or treated with 10

ng/ml TGFa or KGF for 3-4 days. Northern blot analysis was performed using 1 fig ofpoly(A) ' RNA/lane, as described in "Materials and Methods." Expression of transcripts

for EGFR ligands was assessed by reprobing filters multiple times, and uniformity ofmRNA loading was evaluated by probing for glyceraldehyde phosphate dehydrogenase(GAPDH) transcripts. The three TGFa transcripts detected in stimulated wa-l keratino

cytes have approximate lengths of 4.5, 4.7, and 5.2 kilobases. Based on densitometricanalysis and normalization to glyceraldehyde phosphate dehydrogenase, the maximumcombined expression level of the three wa-l TGFa mRNAs (v-rai"" lane) was 6-foldlower than the level of the TGFa transcript in corresponding BALB/c v-r«i"akeratinocyte

cultures. AR, amphiregulin; BC, betacellulin.

nocytes. In control cultures, TGFa measured in cell lysates was at orslightly above the assay background (31 ±4 pg/60-mm dish) in bothBALB/c and wa-l keratinocytes. In contrast, v-raiHa increased the

abundance of cell-associated TGFa in BALB/c keratinocytes to176 ±50 pg/60-mm dish, while the TGFa level in wa-l v-rasHa

keratinocyte lysates was only slightly higher than in control wa-l

keratinocytes (47 ±1.2 versus 34 ±2.1 pg/60 mm dish, respectively)(Fig. 3A).

A similar analysis was performed using v-ras"" primary keratino

cytes from TGFa +/+ and TGFa -/- mice. Like BALB/c keratinocytes, TGFa +/+ primary keratinocytes transduced with v-rasHa

expressed high levels of both secreted (799 ±81 pg/ml) and cell-associated (118 ±29 pg/60-mm dish) TGFa; in contrast, in cultures

of TGFa —/—keratinocytes, TGFa was not increased by \-ras"*

above control levels (Fig. 3ß).CM and cell lysates were also preparedusing established cell lines derived from v-ra.vlla-transduced primarykeratinocytes, as described in "Materials and Methods." The raj-

transformed control cell line a + 2 produced 980.6 ±111 pg/ml ofsecreted TGFa and 420.2 ±116 pg/60-mm dish of cell-associated

TGFa. In contrast, there was no detectable increase in either secretedor cell-associated TGFa in three ras-transformed cell lines derivedfrom TGFa -/- epidermal cultures (Fig. 3C). Together, these dataconfirm that cultured TGFa -/- and wa-l keratinocytes exhibit aprofound defect in v-ra.vHa-induced TGFa expression, supporting

results reported previously in which TGFa expression was measuredin tissues from these mouse strains (23, 42). Combined with the datapresented in Figs. 1 and 2, these results indicate that phenotypicchanges induced by v-rasH* in cultured epidermal keratinocytes,

involving growth regulation as well as marker expression, are notdependent on autocrine stimulation by TGFa.

Expression of Transcripts Encoding Multiple EGFR Ligands IsRegulated Coordinately in Normal and TGFa-deficient Keratinocytes. To address the possibility that another growth factor may becompensating for TGFa in wa-l and TGFa —/—keratinocytes, ex

pression of transcripts encoding other mammalian EGFR ligands wasdetermined by Northern blot analysis of poly(A)+ RNA from controland TGFa-deficient keratinocytes. v-raiHa increased the expression

of TGFa transcripts in both wa-l and BALB/c cultures, although the

induction in BALB/c keratinocytes was substantially higher than inwa-l cultures (Fig. 4). In contrast to TGFa mRNA in normal keratinocytes, which is detected as a single species —4.5kilobases long,three TGFa transcripts were present in RNA from wa-l keratinocytes,with estimated sizes of —4.5,4.7, and 5.2 kilobases (Fig. 4). Thesethree bands were also detected in RNA from wa-l brain and skin (Fig.

nu wa-1

.E e -E5 15 2

CQ C/) Cu

TGFa

PL-7

Fig. 5. Multiple TGFa transcripts are expressed in wa-l tissues in vivo. Northern hlotanalysis was performed using 15 jig of poly(A)+-enriched RNA isolated from the

indicated wa-l or nude (nu) mouse tissues. The filter was reprobed for PL-7 mRNA as acontrol for loading. Based on densitometric scanning and normalization to PL-7, thecombined abundance of the three TGFa transcripts in RNA from wa-1 brain was 42% ofthe level detected in nu mouse brain RNA.

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EGFR LÕGANOS AND EPIDERMAL NEOPLASIA

i _ II __

Ìf> if»C R> C (Oo v o >u > u >

•-TGF-a

AR

HB-EGF

BTCL

GAPDH

Fig. 6. v-raslia coordinately induces transcripts encoding multiple EGFR ligands in

TGFa-null and control keratinocytes in vitro. Primary epidermal keratinocytes fromTGFa null (-/-) and control (+/+) mice were grown under basal conditions ortransduced with \-ras"'. Poly(A)+ RNA from the indicated cultures (3 /j.g/lane) was

analyzed by Northern blotting. The targeting strategy used for generating TGFa-null miceresults in the production of a 6.2-kilobase TGFa transcript in —/—keratinocytes. GAPDH,

glyceraldehyde phosphate dehydrogenase; AR, amphiregulin; BTCL, betacellulin.

5), indicating that the expression of multiple TGFa transcripts is notrestricted to stimulated wa-\ keratinocytes in vitro. In agreement witha previous report (42), the expression of TGFa mRNA in wa-\ brainwas lower than in control brain; however, only the 4.5-kilobase TGFatranscript was detected in the earlier study (see "Discussion").

Stimuli which induce TGFa gene expression also increase the levelof amphiregulin, HB-EGF, and betacellulin transcripts in culturedkeratinocytes (Fig. 4). In addition to v-rasHa, treatment with TGFa or

KGF up-regulated expression of multiple EGFR ligands in BALB/cand wa-\ cultures (Fig. 4), supporting our recent findings suggesting

that KGF influences keratinocyte biology via activation of the EGFRsignaling pathway (43). Up-regulation of multiple EGFR ligands in

stimulated keratinocytes does not involve all members of this growthfactor family, since transcripts encoding EOF were not detected inepidermal cultures under any of the conditions tested (data notshown).

The effects of v-ras"" on EGFR ligand gene expression were alsoexamined in keratinocytes from TGFa +/+ and TGFa —/- mice.

The usual single 4.5-kilobase TGFa mRNA was up-regulated inv-rasHa-transduced TGFa +/+ keratinocytes, whereas a 6.2-kilobase

TGFa transcript, resulting from the introduction of a 1.7-kilobasePGK-neo cassette in the targeted TGFa gene (23), was induced inTGFa null v-rasHa keratinocytes. Furthermore, as observed with wa-land BALB/c keratinocytes, v-rasHa coordinately induced expression

of amphiregulin, HB-EGF, and betacellulin mRNAs in TGFa-null

and control keratinocytes (Fig. 6). Together, these data indicate thattranscripts encoding multiple EGFR ligands are coordinately regulated in normal as well as TGFa-deficient cultured keratinocytes,

providing a potential mechanism whereby loss, or deficiency, of oneEGFR ligand may be supplanted by another.

Control and TGFa-deficient v-ras"" Keratinocytes Produce

Tumors When Grafted onto Athymic Mice. Tumorigenicity wasassessed by grafting v-rasHa keratinocytes as a reconstituted skin onto

nude mouse hosts (5). Control (BALB/c and TGFa +/+) as well asTGFa-deficient (wa-l and TGFa -/-) v-rasHa keratinocytes pro

duced epidermal tumors in skin grafts (Table 1; Fig. 7). In severalexperiments, the appearance of tumors produced by ras-transformedwa-l and TGFa -/- keratinocytes was delayed when compared

with control tumors. Interestingly, tumors produced by primary wa-lv-raiHa keratinocytes exhibited a greater propensity for malignant

conversion than control BALB/c cells (Table 1). In addition, animmortalized wa-l v-rasHa-transformed cell line (wa-lrp; see"Materials and Methods") also exhibited an increased tendency for

malignant conversion, when compared to two control cell lines, SP-1and C57rp, expressing activated ras11*(Table 1; Fig. 7). Since a region

Table 1 Tumorigenicity of TGFa-deficient keratinocytes expressing oncogenic ras " in

nude mouse skin grafis

v-ras a-transduced primary keratinocytes

Latency" Tumor

Experiment Strain (wk) incidence Histology'

- Graft ageat sacrifice

(wk)

wa-l 3-7 4/5 Carcinoma/spindle 13cell tumor (l/4)d

Papilloma/spindle celltumor (1/4)

Skin (2/4)f

BALB/c 3 5/5 Papilloma (3/4)Skin (1/4)

wa-l 2-4 10/10 Papilloma/carcinoma (1/8) 10

Dysplastic papilloma (2/8)Papilloma (4/8)Skin (1/8)

34BALB/cTGFa-/-TGFa

+/+TGFa

-/-TGFa

+/+2-32-32MVNA3/54/42/23/44/5Skin

(2/2)Papilloma

(3/4)Skin(1/4)Papilloma(2/2)Papilloma

(3/4)Skin(1/4)Papilloma(3/4)Skin

(1/4)55Celllines*56wa-lrpSP-1wa-lrp2-52-54-89/1010/109/10Spindle

cell tumor(1/4)Carcinoma(3/4)Papilloma/carcinoma(1/1)Carcinoma

(1/3)1210Carcinoma/spindle cell tumor (2/3)

7 wa-lrp 2-4 9/9 Papilloma/carcinoma (2/8) 5Carcinoma (1/8)Carcinoma/spindle cell tumor (4/8)Carcinoma/spindle cell

tumor/papilloma (1/8)C57rp 4 5/5 PapUloma (5/5)

4 a-1 NA 5/5 Papilloma (4/5) 5Dysplastic papilloma (1/5)

8 a-2 NA 3/4 Spindle cell tumor (3/4) 7Skin (1/4)

wa-lrp NA 3/4 Papilloma/carcinoma (2/4)Spindle cell tumor (1/4)Skin (1/4)SP-1 NA 2/2 Papilloma (2/2)

a Time interval after grafting when tumor(s) first reported.h Maximum number of tumor-bearing animals/total animals.' Certain tumors contained different regions with distinct histopathologies.''Tumor-bearing animals/total animals remaining at completion of experiment.

Significant animal mortality occurred in some experiments.e Presence of relatively normal-appearing skin in graft site. May result from sponta

neous regression of certain benign lesions or poor graft "take" at the outset of the

experiment.^NA, data not available.* wa-lrp, v-rajHa-transformed iva-1 keratinocyte cell line; SP-1, SENCAR papilloma-

derived cell line containing mutated c-ras a; C57rp, v-ras a-transformed C57BL keratinocyte cell line; a-1, a-2, v-rasHa-transformed TGFa -/- keratinocyte cell lines. See"Materials and Methods" for additional details.

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EOFR LÕGANOS AND EPIDERMAL NEOPLASIA

Fig. 7. Control and TGFa-deficient keratino-cyles expressing the v-ra.(Ha oncogene produce tu

mors when grafted onto nude mice. A, B, grossappearance of tumors generated following graftingof control (TGFa +/+) (A) or TGFa-null (-/-)(B) v-ra.sllu keratinocytes onto nude mice, as described in "Materials and Methods." Animals were

photographed 5 weeks after grafting. C-F, histopa-thology. Tumor sections were stained with H & E.Photomicrographs of representative lesions reveal apattern typical for benign squamous papillomas.Major changes were not detected when comparingtumors arising from v-rajH"-transduced TGFa+/+ (C, £)and TGFa -/- (D. F) keratinocytes.G, 7-week-old tumor produced by grafted wa-lrp

cell line. Carcinoma in lower portion of photomicrograph is contiguous to overlying squamouspapilloma. C. D. X 15; E, F, G, X 45.

of papilloma was detected in several wa-lrp malignant tumors In similar experiments performed using TGFa-null keratinocytes,harvested either 5 or 7 weeks after grafting (Table 1), it appears the gross appearance and histology of 5-week-old tumors produced bythat these cells underwent rapid neoplastic progression in vivo and grafting either TGFa +/+ or TGFa -/- v-rasHa keratinocytes waswere not malignant at the time of grafting. typical for benign squamous papillomas (Fig. 7). Detection of neo'

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EOFR LÕGANOS AND EPIDERMAL NEOPLASIA

TG Fa^- IG Foc+/+

Fig. 8. TGFa -/- v-rajH" papillomas are comprised of grafted TGFa-null keratinocytes, and not host keratinocytes recruited into the graft site. Dark-field in situ detection of neo'transcripts in papillomas produced by TGFa -/— v-rajHa keratinocyles. Tumor sections were hybridized to an antisense riboprobe specific for neoT mRNA. A positive signal is detectedin the epithelial component of tumors produced by grafting TGFa -/- keratinocytes but not TGFa control (+/+) keratinocytes. Corresponding bright-field images in lower panelsdemonstrate neoplastic epithelium in both +/+ and -/— grafts. X 160.

transcripts by in situ hybridization confirmed that TGFa —/—v-rasHa papillomas were comprised of grafted TGFa-null kerati

nocytes, and not host keratinocytes recruited into the graft site(Fig. 8). Interestingly, a TGFa-null v-raiHa-transformed cell line,

a-2, produced malignant tumors with a spindle cell histology 7

weeks after grafting (Table 1). Together, these results indicate thatautocrine TGFa is dispensible for the growth of benign as well asmalignant epidermal tumors.

Squamous Papillomas Express Transcripts Encoding MultipleEGFR Ligands. In light of the in vitro findings presented in Figs. 4and 6, Northern blot analysis was performed to determine the expression pattern of EGFR ligand mRNAs in papillomas produced bygrafting v-raiHa-transformed control or TGFa-null keratinocytes.

Consistent with earlier reports using other mouse strains (10, 13),TGFa mRNA was readily detected in total RNA samples preparedfrom TGFa +/+ papillomas; in contrast, the recombinant 6.2-kilo-

base TGFa transcript was expressed in tumors produced by TGFa-/- v-rasHa keratinocytes (Fig. 9). The paucity of native TGFatranscripts in TGFa -/- tumors validates the use of this model

system for studying in vivo effects of autocrine TGFa deficiency onneoplastic keratinocytes. Further analysis of tumor RNA revealed thatamphiregulin, HB-EGF, and betacellulin transcripts are similarly up-regulated in both control and TGFa -/- v-ras"" tumors (Fig. 9).

Intriguingly, the only EGFR ligand transcript detected in poly(A)"1"

RNA isolated from newborn mouse epidermis was that encodingbetacellulin (Fig. 9). Collectively, these data indicate that autocrineTGFa is not required for the development of v-rasHa-induced squa-

mous papillomas and are in keeping with in vitro results demonstrating coordinate up-regulation of multiple EGFR ligands in stimulated

keratinocytes.

DISCUSSION

Utilizing two mouse strains with distinct genetic defects in TGFaexpression, we have demonstrated that formation of tumors comprisedof epidermal keratinocytes expressing the v-rasHa oncogene is not

dependent on autocrine TGFa. TGFa-deficient and control tumorsexpress transcripts encoding amphiregulin, HB-EGF, and betacellulin,and mRNAs encoding these EGFR ligands are coordinately up-regulated in cultured keratinocytes in response to v-rasHa as well as

exogenous TGFa or KGF. These findings raise the possibility thatanother EGFR ligand, or ligands, may contribute to autocrine growthregulation of ras-transformed keratinocytes.

Increased TGFa expression has been demonstrated in a variety ofhuman epithelial tumors, cultured primary and immortalized tumorcells, and normal epithelial cells transformed in vitro (reviewed in

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EGFR LÕGANOSAND EPIDERMAL NEOPLASIA

,Epi.

TGF-a

HB-EGF

GAPDH

BC

GAPDH

keratin 1

Fig. 9. Transcripts encoding multiple EGFR ligands are coordinately up-regulated insquamous papillotnas. Tolal RNA was isolated from 8 tumors generated by graftingcontrol ( + /+) and TGFot-null (~/~) v-ra.sH" keratinocytes onto nude mice, as describedin "Materials and Methods." Lanes 1-4 and 5-8, RNA isolations from tumors generated

in two independent experiments. Each of the paired lanes contains RNA from a separatetumor produced by grafting v-rajHa keratinocytes of the indicated genotype onto nudemice, as described in "Materials and Methods." Thirty /Ag of tumor RNA were loaded/lane, with 1.5 ftg of poly(A) + RNA from newborn mouse epidermis (Epi.). Despite

the extremely low level of glyceraldehyde phosphate dehydrogenase (GAPDH) transcriptsin epidermal mRNA, keratin 1 mRNA is easily detected. AR, amphiregulin; BC,betacellulin.

Refs. 8 and 9). The observation that EGFR levels are frequentlyup-regulated in human carcinomas (reviewed in Refs. 44 and 45)

strengthens the hypothesis that autocrine activation of this signalingpathway is important for tumor growth. Furthermore, TGFa trans-

genie mouse strains are predisposed to the development of neoplasiainvolving the skin (15, 16), breast (46, 47), pancreas (48), and liver(49). Involvement of an autocrine TGFa loop in tumor cell growth hasbeen substantiated using neutralizing antibodies or antisense DNA insome neoplastic cell types in vitro (50-53) and in xenografts of an

ovarian carcinoma cell line (54). However, neutralizing EGFR antibodies are substantially more effective at blocking tumor growth thanligand-specific antibodies (reviewed in Ref. 55), suggesting that up-

regulation of multiple EGF family members, as reported here, mayoccur in other tumor models. In addition, the ability of TGFa toinduce multiple other EGFR ligands (Fig. 4; Ref. 38) raises thepossibility that one of these other growth factors may play a criticalrole in the development of epithelial tumors in TGFa transgenic mice.

The delayed appearance of tumors produced by TGFa-deficient

keratinocytes suggests that although this growth factor is not obligatory for rai-mediated epidermal neoplasia, it may be an important

regulator of tumor growth during certain stages of this process. A

similar conclusion was reached in a study where human TGFa wasoverexpressed in skin grafts of the papilloma-producing SP-1 cell line

(17). On the other hand, a relatively high incidence of carcinomadevelopment was observed in ra.ï-transformed wa-l keratinocyteswhen compared to ras-transformed control cells. This was the case forprimary wa-l keratinocytes as well as an immortalized cell line,wa-lrp (Table 1). Although it is conceivable that the aggressive nature

of these tumors is related to TGFa deficiency, it may also reflectstrain differences in the behavior of keratinocytes from wa-l versus

other mouse strains. Alternatively, since the ABP/Le mouse strainused for wa-l studies is also homozygous for 5 additional recessivemutations (non-agouti, brown, belted, pink eye, and short ear), one ofthese other alíelesmay be influencing the behavior of ras-transformed

keratinocytes. Strain differences can influence premalignant progression,as demonstrated by the high incidence of chemically induced epidermalcarcinomas developing in FVB/N mice when compared to other strains(56). However, the development of spindle cell tumors in TGFa-null a-2

grafts is consistent with the notion that the absence of autocrine TGFapredisposes to neoplastic progression. Additional grafting studies, chemical carcinogenesis experiments using TGFa-null and control mice, andbreeding the wa-l mutation onto a defined genetic background will be

required to critically address this issue.We have detected several intriguing abnormalities in the expression

of TGFa in wa-l keratinocytes and tissues: (a) the induction of TGFamRNA by v-rajHa is markedly lower in wa-l keratinocyte cultures

than control cultures, consistent with previous reports suggesting thatthe wa-l mutation influences the regulation of TGFa gene expression

(23, 42); (b) in contrast to normal mouse TGFa, which is detected asa single 4.5-kilobase transcript, we have identified two additional

TGFa species, 4.7 and 5.2 kilobases long, in RNA isolated fromcultured wa-l keratinocytes and tissues (Figs. 4 and 5); (c) in addition

to the quantitative changes in expression of TGFa mRNA, the level ofimmunoreactive TGFa produced by wa-l v-raiHa keratinocytes ap

pears to be disproportionately low relative to the amount of mRNApresent, suggesting an abnormality at the protein level as well. Ourfindings differ from those reported in a previous study, in which onlya single 4.5-kilobase TGFa transcript was detected in RNA isolatedfrom wa-l brain (42). However, since the probe used in the earlier

report was a partial TGFa cDNA encoding 2.0 kilobases of exon 6, itis conceivable that we are detecting transcript variants hybridizing tofull-length cDNA that are missing this region of the normal TGFatranscript. Studies aimed at identifying the nature of the wa-l muta

tion should provide an explanation for the complex alteration in TGFaexpression observed in these cells.

Our findings suggest that another EGFR ligand may be capable ofsubstituting for TGFa in autocrine growth stimulation of ras-trans-

formed keratinocytes. Consistent with this hypothesis, amphiregulinhas been shown to act as an autocrine growth regulator in raj-

transformed human mammary epithelial cells (57) and a colon carcinoma cell line (58) in vitro. However, the potential autocrine functionof HB-EGF or betacellulin, both of which are up-regulated by v-rasHa

in vitro (Figs. 4 and 6) and in vivo (Fig. 9), has not been previouslyaddressed in neoplastic keratinocytes. We have recently reported thatCM from v-rasHa keratinocyte cultures induces phenotypic changes in

normal cells mimicking those observed in ras-transformed cells.

Moreover, these alterations are blocked using a neutralizing TGFaantibody (11), indicating that TGFa is the principal secreted factorresponsible for v-raiHa effects mediated through the EGFR. The

results presented in this report suggest that in cultures of TGFa —/—v-ra.ïHakeratinocytes, other EGFR ligands may be capable of induc

ing responses previously attributed solely to TGFa. We are currentlyassessing the overall importance of the EGFR signaling pathway in

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normal keratinocyte biology and neoplasia by disrupting EGFR function using antisense cDNA and a dominant-negative mutant (59, 60).

Simultaneous induction of mRNA encoding four EGFR ligands inepidermal keratinocytes raises questions regarding the role of thesegrowth factors, both individually and collectively, in epidermal ho-

meostasis and disease. Despite certain similarities, these growth factors exhibit distinct biochemical differences that may influence theirfunction and localization both in vitro and in vivo; for example,amphiregulin, HB-EGF, and betacellulin bind heparin, whereas ma

ture TO Fa does not (21, 61). Furthermore, although heparin blocksthe mitogenic response of cultured keratinocytes to amphiregulin (62),it enhances HB-EGF-induced proliferation (63) and migration (64)

but appears to have no effect on cell growth stimulated by betacellulin(65). Interaction with heparan sulfate proteoglycans in vivo may havesimilar functional consequences and could also provide a means ofrestricting activity of individual growth factors to specific sites withina tissue, e.g., during wound-healing.

Several studies have explored the expression of EGFR ligands inepidermis and/or cultured keratinocytes. TGFo (66), amphiregulin(62), and HB-EGF (67) are expressed by cultured human epidermal

keratinocytes. In addition, TGFa is present in human epidermis atrelatively low levels (66, 68), and HB-EGF mRNA has been detectedby RT-PCR in RNA isolated from skin (69). Up-regulation of TGFa

and amphiregulin mRNA, as well as TGFa protein, has been described in the hyperproliferative skin disease psoriasis (68, 70, 71),and elevated expression of TGFa protein has been detected in severaltypes of human epidermal tumors (68). Moreover, HB-EGF was

identified in wound fluid obtained from a porcine skin experimentalmodel (63). Expression of the most recently described EOF familymember, betacellulin, has not been reported previously in skin orcultured keratinocytes; however, the preponderance of betacellulinmRNA in newborn mouse epidermis (Fig. 9) suggests that it is a majorEGFR ligand produced by this tissue. Additional studies to determinethe abundance and distribution of EGFR ligands, in both newborn andadult mouse skin, may provide clues into the normal role of thesegrowth factors in cutaneous biology, and help explain the selectivealteration of hair follicle function observed in both wa-\ and TGFa

null mice.

ACKNOWLEDGMENTS

The authors are grateful to Dr. Rolf Kemler for K8 antibody, Dr. WendyWeinberg for providing the C57rp cell line, Drs. Kay Gray Nelson andCaroline Stanwell for PL-7 cDNA, and Dr. Glen Merlino for critical reading of

the manuscript. The technical assistance of David Morgan during skin graftingstudies was greatly appreciated, as was animal breeding performed by MarthaShaver, Karen Baxley, Susan Kirchhoff, and Linda Sly (Biocon, Inc.,Rockville, MD).

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on July 18, 2018. © 1995 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

1995;55:1883-1893. Cancer Res   Andrzej A. Dlugosz, Christina Cheng, Erin K. Williams, et al.   Alternate Epidermal Growth Factor Receptor Ligands

-induced Epidermal Neoplasia: Potential Involvement ofHaras Is Dispensible for v-αAutocrine Transforming Growth Factor

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