©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Epiregulin
A NOVEL EPIDERMAL GROWTH FACTOR WITH MITOGENIC ACTIVITY FOR RAT PRIMARY HEPATOCYTES (*)

(Received for publication, November 1, 1994; and in revised form, January 1, 1995)

Hitoshi Toyoda Toshi Komurasaki (§) Daisuke Uchida Yasuko Takayama Toshiaki Isobe (1) Tuneo Okuyama (1) Kazunori Hanada

From the Department of Applied Biology, Research Center, Taisho Pharmaceutical Co., Ltd., 1-403, Yoshino-cho, Ohmiya, Saitama 330, Japan Department of Chemistry, Faculty of Science, Tokyo Metropolitan University, Hachioji-shi, Tokyo 192-03, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PrOCEDURE
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Epiregulin, a novel epidermal growth factor (EGF)-related growth regulating peptide, was purified from conditioned medium of the mouse fibroblast-derived tumor cell line NIH3T3/clone T7. It was a 46-amino-acid single chain polypeptide, and its amino acid sequence exhibited 24-50% amino acid sequence identity with sequences of other EGF-related growth factors. Epiregulin exhibited bifunctional regulatory properties: it inhibited the growth of several epithelial tumor cells and stimulated the growth of fibroblasts and various other types of cells. Epiregulin bound to the EGF receptors of epidermoid carcinoma A431 cells much more weakly than did EGF, but was nevertheless much more potent than EGF as a mitogen for rat primary hepatocytes and Balb/c 3T3 A31 fibroblasts. These findings suggest that epiregulin plays important roles in regulating the growth of epithelial cells and fibroblasts by binding to receptors for EGF-related ligands.


INTRODUCTION

Cellular growth and differentiation are modulated by a number of peptide growth factors. Accumulated evidence supports that growth control cascade involves growth factors and their receptors in both tumor and non-tumor cells(1) . According to the autocrine hypothesis, many tumor cells synthesize and release excess amounts of certain growth factors, resulting in the abnormal proliferation of tumor cells due to mitotic stimulation by the growth factors that they produced(2, 3) . On the other hand, several growth inhibitors have been purified so far(4, 5, 6, 7, 8) . Loss or reduction of the susceptibility of cells to growth inhibitors is also argued about the mechanism of malignant transformation(3) . Cellular proliferation is regulated by the interplay of growth regulators in both positive and negative fashions. In addition, certain tumor cell growth inhibitors also act as factors that stimulate the growth or induce the differentiation of other types of cells(4, 6, 7, 8, 9) . During the screening of conditioned media of cultured cells, we detected an activity which induced the morphological changes in the human epitheloid carcinoma cell line HeLa, in the conditioned medium of NIH3T3/clone T7, a cell clone of mouse fibroblast NIH/3T3 that was adapted in protein-free medium and then acquired tumorigenicity. The conditioned medium also inhibited the growth of several tumor cells. Here we report the purification, structure, and biological activities of this novel growth regulator, namely, epiregulin, which is a new member of the epidermal growth factor (EGF) (^1)family.


EXPERIMENTAL PrOCEDURE

Cells and Materials

HeLa, A431, A549, and Balb/3T3 clone A31 cells were purchased from Dainihon Pharmaceutical Co., Osaka. Recombinant human hepatocyte growth factor was purchased from Toyobo Co., Tokyo. Recombinant mouse EGF and recombinant rat TGF-alpha were obtained from Collaborative Research and Bachem Fine Chemicals, respectively.

Purification

NIH 3T3/clone T7 cells were maintained in DF medium (Dulbecco's modified Eagle's: Ham's medium F12 = 1:1) supplemented with 10% fetal bovine serum (FBS). After cells had grown to confluence, the medium was replaced with DF medium. The culture medium was collected twice per week. Sixty liters of the conditioned medium were concentrated about 20-fold with a Filtron ultrafiltration system using membrane with a 1-kDa cut-off molecular size (Fujifilter Co., Tokyo) and precipitated by 90% saturation of ammonium sulfate. The precipitates were dialyzed against 20 mM Tris-HCl, pH 7.5, applied to a Q-Sepharose (Pharmacia) column (5 times 16 cm) equilibrated with the same buffer, and then the column was washed extensively with the buffer. Half of the activity was eluted into the flow-through and wash fractions. These active fractions were adjusted to pH 5.0 with acetic acid and applied to a S-Sepharose (Pharmacia) column (2.5 times 6 cm) equilibrated with 20 mM acetate buffer, pH 5.0. The bound materials were eluted with 20 mM Tris-HCl, pH 7.5. The eluate was adjusted to pH 6.0 with acetic acid and passed through a hydroxyapatite column (7.5 times 100 mm, Pentax) equilibrated with 20 mM acetate buffer, pH 6.0. The flow-through fraction was then adjusted to pH 5.0 with acetic acid, and subjected to a CM-3SW high performance liquid chromatography (HPLC) column (7.5 times 7.5 mm, Toso Co., Tokyo). The eluate was developed with a linear gradient of 0-0.2 M NaCl in 20 mM acetate buffer, pH 5.0, and 5% acetonitrile at a flow rate of 1 ml/min. The activity was eluted in two peaks at 80 mM (P-I) and 100 mM NaCl (P-II). The fractions in each peak were subjected to a phenyl 5PWRP reverse-phase (RP)-HPLC column (4.6 times 7.5 mm, Toso Co., Tokyo) equilibrated with 5% acetonitrile in 5 mM phosphate buffer, pH 7.4, and eluted with 20% acetonitrile followed by a linear gradient of 20-40% acetonitrile in 5 mM phosphate buffer, pH 7.0, at a flow rate of 0.5 ml/min. The activities were eluted by 25% (P-I) and 22% acetonitrile (P-II) in 5 mM phosphate buffer, pH 7.4, in the respective peak fraction. The elution profiles of the activities coincided with those of the absorbance at 210 nm. The active fractions were lyophilized and reconstituted with distilled water.

Assay of Morphological Changes in Cultured Cells

HeLa cells were plated into 48-well plates at a density of 2.5 times 10^4 cells/well in 0.25 ml of DF medium supplemented with 10% FBS. After 24 h, the medium was replaced with DF medium, and 0.5-20-µl aliquots of purified fractions were added to each well. After 15-48 h of incubation, the activities required to induce the morphological change in HeLa cells round shape were assayed microscopically and compared with those for control culture.

Cell Proliferation Assay

HeLa, A431, A549, or Balb/3T3 clone A31 cells were plated into 6-well plates at a density of 5 times 10^3 cells/well in 2 ml of DF medium supplemented with 10% FBS. After 24 h, the medium was replaced with DF medium supplemented with 2% FBS, and cells were treated with various growth factors. Five days later, surviving cells were counted using the trypan blue exclusion method.

Rat hepatocytes were isolated as described(41) . Hepatocytes were seeded in collagen-coated 24-well plates at a density of 2.5 times 10^5 cells/well in 1 ml of DF medium supplemented with 10% FBS. After 6 h, the medium was replaced with 0.5 ml of DF medium supplemented with 5% FBS, and cells were incubated with epiregulin or other growth factors for 48 h. [^3H]Thymidine (Amersham) was added to 2.5 µCi/ml during the final 16-h period of incubation. Cells were harvested by trypsinization, and the radioactivities were counted with a Betaplate system (Pharmacia).

Human aortic smooth muscle cells (Kurabo Co., Osaka) were plated, as were rat hepatocyte culture, at a density of 5 times 10^3 cells/well. After 24 h, the medium was replaced with DF medium supplemented with 2% FBS, and cells were incubated with epiregulin or other growth factors for 48 h. Incorporation of [^3H]thymidine into the cellular DNA was determined. In these assays, two independent experiments on triplicate plates were carried out.

Analysis of Amino Acid Sequence and Composition

Two active peaks, for P-I and P-II, obtained from RP-HPLC were desalted by RP-HPLC on a phenyl 5PWRP column with a linear gradient of 5-50% acetonitrile in 0.1% trifluoroacetic acid at a flow rate of 1 ml/min. Approximately 5 µg of purified epiregulin was reduced and S-pyridylethylated in the presence of 6 M guanidium chloride as described(42) , and the S-pyridylethylated peptides were desalted with RP-HPLC. One-fifth of the recovered preparations were subjected to microsequencing. Three-tenths of the recovered preparations were dried under a gentle stream of nitrogen gas, resuspended in 100 µl of 70% formic acid containing 1 mg of cyanogen bromide, and incubated for 20 h at room temperature. The cyanogen bromide-treated peptide fragments were purified by RP-HPLC on a C4 column (4.6 times 100 mm, Vydac) with a linear gradient of 10-50% acetonitrile in 0.1% trifluoroacetic acid at a flow rate of 1 ml/min. Sequence analysis was performed with an automated amino acid sequencer (model 477A, Applied Biosystems) equipped with an on-line model 120A phenylthiohydantoin amino acid analyzer. Amino acid composition was determined with an automated high performance amino acid analyzer (model 800; Jasco, Tokyo) equipped with a post-column derivatization system with o-phthalaldehyde. Samples were hydrolyzed with 6 M HCl containing 5% (v/v) phenol at 110 °C for 24 h in evacuated sealed tubes. The concentration of epiregulin was determined by amino acid analysis.

Binding to EGF Receptor

The binding assay was performed as described (43) with slight modifications. A431 cells were grown to confluence in DF medium supplemented with 10% FBS in 24-well plates. The cells were washed twice with cold phosphate-buffered saline and twice with cold binding buffer of DF medium containing 25 mM HEPES buffer, pH 7.5, and 0.1% bovine serum albumin. Binding was performed at 4 °C with 2 ng/well of mouse I-EGF (10^5 cpm, Amersham) with varying quantities of unlabeled epiregulin, mouse EGF, or rat TGF-alpha. Nonspecific binding was determined in the presence of an excess amount (2 µg/well) of unlabeled EGF. Cells were incubated for 4 h, washed three times with cold binding buffer, and then lysed with 0.4 ml of 1 N NaOH for 30 min at 37 °C. Radioactivities of cell lysates were determined by a WALLAC model 1470 WIZARD -counter (Pharmacia). The nonspecific binding was about 10% of the total amount of radioactivity bound to cells. Two independent experiments on triplicate plates were carried out.

Antibody against Epiregulin

Rabbits were immunized with 500 µg of chemically synthesized epiregulin. (^2)Emulsions of antigen and complete Freund's adjuvant were injected eight times subcutaneously at intervals of 1 or 2 weeks. The serum was precipitated with 40% saturation of ammonium sulfate. The precipitate was dialyzed against 50 mM phosphate buffer, pH 7.4, containing 0.15 M NaCl and applied to a Protein A-Superose column (1 times 2 cm; Pharmacia). The column was eluted with 20 mM glycine-HCl buffer, pH 3.0, and the eluate was immediately neutralized with 1 M Tris to about pH 7.4.

Western Blot Analysis

Five ml of conditioned medium was incubated with 2.5 µg/ml of anti-epiregulin rabbit IgG for 1 h, and then 20 µl of Protein G-Sepharose (50%) was added to the reaction mixture. After incubation for 15 h, the pellets were washed five times with 20 mM Tris-HCl, pH 7.4, containing 0.5% Triton X-100 and 0.15 M NaCl. The precipitates were suspended in 20 µl of SDS-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer. The supernatants were resolved by SDS-PAGE followed and then electrotransferred onto Immobilon-P membrane (Millipore). The membrane was soaked with 1 µg/ml of anti-epiregulin rabbit IgG containing 10% horse serum, 10% Block Ace (Dainihon Pharmaceutical Co., Osaka), and 0.1% bovine serum albumin for 1 h at room temperature. After the membrane was washed with washing buffer, it was incubated with peroxidase-conjugated anti-rabbit Ig (Fab)`(2) for 1 h at room temperature and washed with washing buffer. The detection was carried out using an ECL kit (Amersham).


RESULTS

Purification

Conditioned medium of the mouse tumor cell line NIH3T3/clone T7 induced changes in the morphology of HeLa cells: the cells became round in the serum-free medium and spindle-shaped in the serum-containing medium after incubation for about 15 h (Fig. 1). Similar morphological changes were found when purified epiregulin was added to HeLa cells. The purification procedure is described under ``Experimental Procedures'' in detail. The activity was eluted in two peaks, as P-I and P-II. The samples obtained from P-I and P-II peak fractions were purified to apparent homogeneity by these purification procedures, since they migrated as single bands of about 5 kDa under reducing and non-reducing conditions on 20% SDS-PAGE) (Fig. 2A).


Figure 1: Morpholical changes in Hela cells induces by epiregulin. Hela cells were cultured in the absence (A) or the presence (B) of 100 ng/ml epiregulin for 15 h in serum-free medium.




Figure 2: Identification of epiregulin. A, SDS-PAGE of purified epiregulin. Purified preparation of epiregulin was analyzed on a 20% SDS-polyacrylamide gel and stained with silver. Lane 1, molecular size marker (46 kDa, ovalbumin; 30 kDa, carbonic anhydrase; 21.5 kDa, trypsin inhibitor; 14.3 kDa, lysozyme; 6.5 kDa; aprotinin; 3.4 kDa, insulin chain A; Amersham); lane 2, 150 ng of epiregulin. B, immunoblot detection of epiregulin in the conditioned medium of NIH/3T3 clone T7 cells. The samples were subjected to Western blot analysis. Lane 1, 40 ng of epiregulin; lane 2, 40 ng of epiregulin was immunoprecipitated with anti-epiregulin rabbit IgG; lane 3, 5 ml of conditioned medium was immunoprecipitated with anti-epiregulin rabbit IgG; lane 4, 5 ml of conditioned medium was immunoprecipitated with normal rabbit IgG. The arrowhead indicates the mobility of epiregulin on SDS-PAGE.



Table 1summarizes the results of purification. Approximately 5 and 10 µg of epiregulin, respectively, were recovered in P-I and P-II from 60 liters of the conditioned medium. The overall purification was 13,000-fold, and the recoveries were 1.6 and 3.0%, for P-I and P-II, respectively. The large scale purification was carried out three times. The ratio of recoveries in the two fractions varied in each preparation of the conditioned medium. Two µg of P-I and very small amounts of P-II were obtained from 40 liters; while 5 µg of P-I and 5 µg of P-II were obtained from 100 liters of the conditioned medium.



Epiregulin was immunoprecipitated from the conditioned medium of NIH3T3/clone T7 culture with anti-epiregulin polyclonal antibody and subjected to Western blot analysis (Fig. 2B). The immunoreacted band migrated in similar fashion to the chemically synthesized epiregulin.^2 The upper intense bands were nonspecific and were presumably rabbit IgG, since they were also observed in the lanes in which synthetic epiregulin was applied and in those in which normal rabbit IgG was used for immunoprecipitation instead of anti-epiregulin rabbit IgG. Ten µg of anti-epiregulin rabbit IgG did not recognize the plate coated with 500 ng of mouse EGF in the enzyme-linked immuno-solid assay (data not shown).

Amino Acid Sequence of Epiregulin

The primary structure of epiregulin was determined (Fig. 3). Although the active fractions were eluted in two peaks, as P-I and P-II, on CM-3SW and phenyl-5PWRP columns, the amino acid sequences of the fractions eluted in the two peaks were identical. Since incompletely cleaved fragments containing methionine residue were obtained after cleavage with cyanogen bromide, these P-I and P-II peaks may carry modified amino acid residues such as oxidized methionine. Epiregulin was a single chain polypeptide of 46 amino acid residues with an estimated molecular mass of 5,400 Da, and carried no potential N-linked glycosylation sites. In a search for homology of amino acid sequences in the data base of the Protein International Resource, epiregulin was found to be a new peptide and to belong to the EGF family, each member of which shared six conserved cysteine residues at similar spacings (Fig. 4). In addition to the cysteine residues, tryptophan at 13, histidine at 16, glycine at 17, tyrosine at 36, glycine at 38, arginine at 40, and leucine at 46 were highly conserved in epiregulin and the other members of the EGF family. In particular, the leucine at 46 is the key residue of EGF and TGF-alpha for mitogenic and receptor binding activities(10, 11, 12) . The amino acid sequence of epiregulin was between 24 and 50% identical to that of each member of the EGF family (Fig. 4). The mature forms of certain members of the EGF family, including amphiregulin(13) , heparin-binding EGF-like growth factor(14) , heregulin/neu differentiation factor(15, 16) , and betacellulin(17) , carry additional N-terminal sequences followed by the regions of EGF-related amino acid sequences, but epiregulin did not. Thus, epiregulin is, along with EGF and TGF-alpha, one of the smaller polypeptides in the EGF family.


Figure 3: Amino acid sequence of epiregulin. The solid line shows the peptide fragments (B-I-III) obtained by the cyanogen bromide cleavage of purified epiregulin. The assignments of sequence for residues are indicated by arrows. An analysis of the amino acid composition of about 30 pmol of purified epiregulin supported this sequence.




Figure 4: Sequence comparison of epiregulin and EGF-related polypeptides. Dashes indicate gaps introduced for better alignments. The EGF-related peptides are numbered relative to the N termini of their mature forms on the left of each line. The sequence of heregulin (HRG) begins only with amino acid 177 of proHRG-alpha(15) . Boxed residues indicate amino acids identical to those of epiregulin. Numbers in parentheses indicate percent homology with epiregulin. The sequences are those of vaccinia virus growth factor (VGF) (36, 37) , betacellulin (BTC)(17) , TGF-alpha(38) , EGF(39) , heparin binding EGF-like growth factor (HB-EGF)(40) , and amphiregulin (AR)(13) . The amino acid sequence of epiregulin was deposited in Japanese International Protein Information (accession number JT0747).



Biological Properties

Epiregulin inhibited the growth of human epithelial tumor cells including HeLa, A431, and lung carcinoma A549 cells in a dose-dependent fashion in both serum-free and serum-containing media (Fig. 5). The growth rates of these cell lines were decreased to approximately 35-70% of those of the untreated cultures, when they were incubated with 50 ng/ml epiregulin for 5 days in the medium supplemented with 2% serum. Epiregulin had no effects on the growth of human leukemia cells including promyelocytic leukemia HL-60 and chronic myelogenous leukemia K562 cells up to 100 ng/ml (data not shown).


Figure 5: Growth inhibitory activities of epiregulin for tumor cell lines. HeLa (cross-hatched bar), A549 (hatched bar), and A431 (stippled bar) cells were cultured with various concentrations of epiregulin for 5 days. Values are cell number relative to those for the control culture to which epiregulin was not added.



On the other hand, epiregulin, as well as TGF-alpha and recombinant hepatocyte growth factor, stimulated DNA synthesis of rat primary hepatocytes at concentrations of 0.8-20 ng/ml, while EGF induced only an approximately 7-fold increase at similar concentrations (Fig. 6A). The profiles of dose-dependent stimulatory activity by epiregulin were similar to those for TGF-alpha, but not to those for EGF. Maximal stimulation was observed at 10 ng/ml of EGF and at 20 ng/ml of TGF-alpha, but the stimulation was increased by epiregulin up to concentrations above 20 ng/ml. Therefore, epiregulin is one of the most potent mitogens known for hepatocytes. The stimulatory effect of epiregulin was additive with those of hepatocyte growth factor or TGF-alpha at concentrations of 4-20 ng/ml. On addition of both EGF and epiregulin to the culture medium, the growth stimulatory effect was lower than that with epiregulin alone. (^3)


Figure 6: Growth stimulatory activities of epiregulin. A, stimulation of DNA synthesis in the primary culture of rat hepatocytes. B, stimulation of proliferation of Balb/3T3 clone A31 cells. C, stimulation of DNA synthesis of human vascular smooth muscle cells. Cells were incubated with various concentrations of epiregulin (bullet), human hepatocyte growth factor (circle), mouse EGF (up triangle), or rat TGF-alpha (box). Stimulation index values are relative to those of control cultures. One stimulation index unit = 4.1 times 10^3 cpm (A), 2.1 times 10^4 cells (B), 8.5 times 10^2 cpm (C). The methods are described under ``Experimental Procedures.''



Epiregulin displayed higher potency as a mitogen for mouse fibroblast Balb/3T3 A31 cells at concentrations above 10 ng/ml than did EGF (Fig. 6B). Epiregulin stimulated the proliferation of Balb/3T3 A31 cells 13-fold at 10 and 18-fold at 100 ng/ml, while EGF induced only an approximately 9-fold increase at these concentrations. However, the stimulation by epiregulin was less potent than EGF at concentrations less than 10 ng/ml.

Epiregulin also stimulated DNA synthesis on human aortic smooth muscle cells (Fig. 6C). The concentrations required for maximum stimulation by epiregulin was higher than those by EGF and TGF-alpha. On the other hand, epiregulin had no significant effects on the DNA synthesis of human endothelial cells from the aortic and the umbilical vein, nor did EGF or TGF-alpha (data not shown).

Binding of epiregulin to the EGF receptor was demonstrated. Although 50 ng/ml unlabeled EGF and TGF-alpha completely competed out binding of I-EGF to A431 cells, the purified epiregulin exhibited only about 20% competition at the same concentration. To ascertain if epiregulin can interact with EGF receptors on A431 cells, the effect of synthetic epiregulin in the binding of I-EGF to A431 cells was investigated (Fig. 7). Unlabeled synthetic epiregulin competed withI-EGF binding to A431 cells in a dose-dependent fashion at concentrations above 10 ng/ml to 1 µg/ml. Fifty % inhibition of binding of I-EGF was observed at 122 ng/ml of epiregulin, but at 8 ng/ml of EGF. These findings suggest that epiregulin has a lower affinity for EGF receptors on A431 cells than does EGF.


Figure 7: Competition with I-EGF in binding to A431 cells. I-EGF (2 ng, 10^5 cpm) was added to the confluent A431 cultures with various amounts of epiregulin (bullet), mouse EGF (up triangle), or rat TGF-alpha (box).




DISCUSSION

Epiregulin was originally identified as a tumor growth inhibitor which induced morphological changes in HeLa cells. In addition to the well-characterized EGF and TGF-alpha, several new members of the EGF family including amphiregulin(13) , heparin-binding EGF-like growth factor(14) , heregulin/neu differentiation factor(15, 16) , and betacellulin (17) have been purified. Epiregulin, as well as, other members of the EGF family, mediated signals stimulating or inhibiting the proliferation of target cells of various types. However, the biological characteristics of epiregulin differed from those of other members of the EGF family in the following respects: 1) epiregulin was more potent than EGF as a mitogen for rat primary hepatocytes at concentrations above 3 ng/ml and for Balb/3T3 A31 cells at concentrations above 10 ng/ml, but less potent as a mitogen for human aortic smooth muscle cells. 2) All of the recently identified members of the EGF family exhibit the ability to bind to heparin-Sepharose columns(13, 14, 15, 16, 17) . However, epiregulin did not bind to a heparin-Sepharose column even in the absence of NaCl in 20 mM Tris-HCl buffer at pH 7.4 (data not shown). Epiregulin, as well as EGF and TGF-alpha, appears to have no binding sites for heparin. 3) A purified preparation of epiregulin and synthetic epiregulin both exhibited much weaker competition than EGF with I-EGF in the binding to A431 cells in which the EGF receptor was overexpressed. Epiregulin, as well as amphiregulin, may bind to the EGF receptor with low affinity(13) . Alternatively, the other receptors which bind EGF-related ligads, HER2/erbB2(18, 19, 20) , HER3/erbB3(21, 22) , and HER4/erbB4(23) , may serve as receptors for epiregulin.

Epiregulin was a more potent mitogen for rat hepatocytes than was EGF. TGF-alpha (24) and heparin-binding EGF-like growth factor (25) each act as potent mitogens for primary cultures of rat hepatocytes by virtue of exerting, respectively, autocrine and paracrine effects via the same high affinity EGF receptor. Hepatocyte growth factor, the most potent hepatotrophic factor (26) yet known, acts in endocrine and paracrine fashion during regeneration of the liver(27, 28) . Epiregulin stimulated DNA synthesis in rat hepatocytes, as did hepatocyte growth factor and TGF-alpha as well. Thus, epiregulin may together with these hepatotrophic factors play an important role in the regeneration of the liver.

Epiregulin was derived from the tumor cell line NIH/3T3 clone T7, which is highly tumorigenic in athymic mice. TGF-alpha and amphiregulin are originally derived from the conditioned media of murine sarcoma virus-transformed cells and the phorbol 12-myristate 13-acetate-treated human mammary adenocarcinoma MCF7 cells, respectively(29, 30, 31, 32) . They act as autocrine growth factors for certain tumor cell lines(31, 32) . In transgenic animals carrying the expression vector for TGF-alpha, a variety of neoplastic lesions are detected, the type of which depends on the strain of mouse and the promoter of the vector(33, 34, 35) . The possibility remains that epiregulin as well as TGF-alpha and amphiregulin may play roles in the initiation or progression of tumors.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 81-48-663-1111; Fax: 81-48-652-7254.

(^1)
The abbreviations used are: EGF, epidermal growth factor; TGF-alpha, transforming growth factor-alpha; PAGE, polyacrylamide gel electrophoresis; HPLC, high performance liquid chromatography; FBS, fetal bovine serum; RP, reverse phase; cpm, counts/min.

(^2)
D. Uchida, S. Funakoshi, N. Fujii, H. Toyoda, and T. Komurasaki, manuscript in preparation.

(^3)
H. Toyoda and T. Komurasaki, unpublished data.


ACKNOWLEDGEMENTS

We thank Y. Mikami, and Y. Sakurai for their technical assistance. We also thank our colleagues in the Biotechnology Group in Taisho Pharmaceutical Co. for their valuable discussions.


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