transforming growth factor-a production and autoinduction ... · densitometer (gs-3000)....

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[CANCER RESEARCH 53. I630-I6.16. April 1. ITO| Transforming Growth Factor-a Production and Autoinduction in a Colorectal Carcinoma Cell Line (DiFi) with an Amplified Epidermal Growth Factor Receptor Gene1 Scema Untawale, Mark A. Zorbas, Clague P. Hodgson, Robert J. Coffey, Gary E. Gallick, Susan M. North, David M. Wüdrick,Matilde Olive, Mark Blick, Lynn C. Yeoman, and Bruce M. Boman2 on Cancer Center. Creighton University School of Medicine. Omaha. Nebraska 68178 IB. M. B.. D. M. W.. C. P. H.f; Department of Tumor Biology. The Universitv of Texan M. D. Anderson Cancer Center. Houston. Texas 77030 ¡G. E. G.. S. M. N.]: Department of Human Oncologv K4/3I9, University of Wisconsin Hospital, University' of Wisconsin Clinical Cancer Center. Madison. Wisconsin 53792 ¡S. U.¡:Section of Gastrointestinal Oncolog\ and Digestive Diseases. The University of Texas M. D. Anderson Cancer Center. Houston. Texas 77030 ¡M. O.I: Molecular Analysis. Inc.. Houston. Texas 77054 ¡M. fi./. Department of Pharmacology. Baylor College of Medicine, Houston. Texas 77030 ¡M. A. Z.. L C. Y./: and Vanderbill University School of Medicine. Departments of Medicine and Cell Biology. Nashville. Tennessee 37232 IR. J. CI ABSTRACT The DiFi colorectal carcinoma cell line, derived From a patient with familial adenomatous polvposis. was examined for gene expression and production of the autocrine growth factor transforming growth factor a (TGF-«) and for epidermal growth factor receptor (EGFR) gene expres sion and gene copy number. DiFi cells expressed TGF-a transcripts as identified on Northern (RNA) blots. Addition of TGF-a ( 10 ng/ml) or EOF 110 ng/ml) to DiFi cell cultures (lacking I <.1 or serum) up-regulated DiFi cell basal T(iF-» mRNA levels, suggesting that autoinduction of TGF-a occurs in these cells. DiFi cell cultures in log phase growth secreted mea surable amounts of TGF-u (347 pg/10* cells/24 h) into their culture me dium, as determined by radioimmunoassay. DiFi cells showed strong over- expression of the EGFR gene on Northern blots relative to three other colon cancer cell lines examined. Immunoperoxidase staining showed en hanced I I.I K expression in a cell subpopulation among the original (un cultured) ascitic fluid cells from which the DiFi cell line was established. This cell subpopulation was observed to expand dramatically between passages 1 and 25. Immune complex kinase assay of DiFi cells showed that EGFR were functional as determined by their ability to autophosphory- late. The EGFR gene in these cells was not found to be rearranged or genetically altered using Southern blot analysis. Dot blot analysis of DiFi cell DNA revealed EGFR gene amplification in the range of 60-80 copies/ cell, which is approximately twice the copy number seen in A-431 epider- moid carcinoma cells. To our knowledge DiFi cells represent the first example of EGFR gene amplification in a colorectal adenocarcinoma. Because Ilili colorectal cancer cells uniquely show production and auto- induction of TGF-a in addition to amplification and overexpression of the EGFR gene, these cells represent a valuable tool for studying the role(s) of the EGFR in the regulation of tumor cell growth. INTRODUCTION EOF' is a polypeptide growth factor produced by many normal human tissues and is present in normal serum. TGF-a, a homologue of EOF. is produced by a variety of malignant and normal cell types. Both TGF-a and EOF are known to bind to the same cell surface receptor, the EGFR. and they produce a similar biological response (reviewed in Refs. I and 2). Received 9/8/92; accepted 1/14/93. 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 with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Support for these studies was provided by Health Future Foundation Funds. Creigh- ton University Department of Medicine Research Fund. The University of Texas M. D. Anderson Cancer Center New Program Funds, and NIH grant CA5039I (L. C. Y.). Preliminary data were presented at the proceedings of the Fourth International Symposium on Colorectal Cancer. Kobe. Japan. November 1989. 2 To whom requests for reprints should be addressed, at Creighton Cancer Center. Creighton University School of Medicine. 24th and California Street. Omaha. NE 68178. 1The abbreviations used are: EGF. epidermal growth factor; TGF-a, transforming growth factor-a; EGFR. epidermal growth factor receptor(s); RIA, radioimmunoassay: ATCC, American Type Culture Collection: FBS. fetal bovine serum; SSC. standard sodium citrate: 10F medium, McCoy's 5A medium (GIBCO) containing UK? FBS; SF-EGF medium. McCoy's 5A medium containing 4 ug/ml transferrin and 20 ug/ml insulin but lacking serum and EGF: DMSO. dimethyl sulfoxide; TPA, 12-O-tetrade- canoylphorbol-l3-acetate; cDNA, complementary DNA. Alterations in EGFR number or structure in a given cell may influence the growth properties of that cell. For example, when NIH 3T3 cells were transfected with a vector containing the full-length EGFR gene (which induces EGFR overproduction), they became fully transformed (3). Additionally, in subclones of A-431 cells having different degrees of EGFR gene amplification and expression, the level of EGFR observed is reported to be proportional to the ability of the cells to grow both in vivo and in vitro (4). The oncogenic prop erties of \-erhB. which encodes a truncated version of the EGFR (5). indicate that an altered EGFR can directly lead to tumorigenesis. Overproduction of EGFR has been reponed for squamous cell carcinomas (lung, skin, oral cavity, and esophagus), brain tumors (glioblastomas and meningiomas). breast adenocarcinomas (90%), and colon adenocarcinomas (moderately well differentiated but not poorly differentiated) (6-13). In some tumors, the overproduction of EGFR is correlated with amplification of the number of copies of the EGFR gene (7. 10. II). In gliomas, the amplified EGFR gene is frequently rearranged, suggesting that this gene mutation may be associated with development of the tumor ( 10). Yet. in the majority of cancers that overproduce EGFR, such overproduction appears to oc cur through mechanisms other than gene amplification. An analysis of 101 cancers, including carcinomas, sarcomas, lymphomas, and leu- kemias. showed that only one squamous cell carcinoma had amplifi cation of the EGFR gene (14). Whereas some primary colonie ade nocarcinomas and colon carcinoma cell lines showed enhanced EGFR expression, none displayed gene amplification (12, 14, 15). The initial purpose of our study was to determine EGFR gene copy number and expression (mRNA) in the DiFi colorectal carcinoma cell line, which was established from a patient with familial adenomatous polyposis (16). Because these studies revealed dramatic EGFR gene amplification/overexpression in DiFi cells ( 17), we conducted further studies on cell surface EGFR expression, EGFR kinase activity. TGF-a production, and potential TGF-a autoinduction in these cells to determine their value in understanding the role of the EGFR in human colon tumor cell growth. MATERIALS AND METHODS Cell Culture. DiFi cells and SW403 colon cancer cells (ATCC CCL 230) were routinely cultured (without udded EGF or TGF-a) in a 50:50 mixture of L-15:Dulbecco's modified Eagle's culture medium (both from GIBCO. Grand Island. NY) containing lO'/r FBS plus insulin (5 ug/ml; Sigma Chemical Company, St. Louis, MO), transferrin (5 ug/ml; Sigma), and sodium selenite (5 ng/ml: Collaborative Research. Waltham. MA) and plated into standard 75-cm- plastic tissue culture flasks or 850-cnr plastic roller bottles (Coming Glass, Corning. NY). The three remaining SW lines. SW480. SW620 (ATCC CCL 228 and CCL 227). and SW742 (from Dr. Benjamin Drewinko, The University of Texas M. D. Anderson Cancer Center. Houston. TX), were grown in L-15 medium (modified) containing i-glutamine. supplemented with \07c FBS. insulin (5 ug/ml). and glutathione (16 ug/ml). A-431 cells (ATCC CRL 1555) used for EGFR gene amplification comparison with DiFi cells were maintained in Dulhecco's modified Eagle's medium with 4.5 g/liter glucose 1630 Research. on November 2, 2020. © 1993 American Association for Cancer cancerres.aacrjournals.org Downloaded from

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Page 1: Transforming Growth Factor-a Production and Autoinduction ... · densitometer (GS-3000). Immunocytochemical Staining. Uncultured metastatic cells from ascitic fluid aspirated from

[CANCER RESEARCH 53. I630-I6.16. April 1. ITO|

Transforming Growth Factor-a Production and Autoinduction in a Colorectal

Carcinoma Cell Line (DiFi) with an Amplified Epidermal Growth FactorReceptor Gene1

Scema Untawale, Mark A. Zorbas, Clague P. Hodgson, Robert J. Coffey, Gary E. Gallick, Susan M. North,David M. Wüdrick,Matilde Olive, Mark Blick, Lynn C. Yeoman, and Bruce M. Boman2

on Cancer Center. Creighton University School of Medicine. Omaha. Nebraska 68178 IB. M. B.. D. M. W.. C. P. H.f; Department of Tumor Biology. The Universitv ofTexan M. D. Anderson Cancer Center. Houston. Texas 77030 ¡G. E. G.. S. M. N.]: Department of Human Oncologv K4/3I9, University of Wisconsin Hospital, University' of

Wisconsin Clinical Cancer Center. Madison. Wisconsin 53792 ¡S. U.¡:Section of Gastrointestinal Oncolog\ and Digestive Diseases. The University of Texas M. D. AndersonCancer Center. Houston. Texas 77030 ¡M. O.I: Molecular Analysis. Inc.. Houston. Texas 77054 ¡M. fi./. Department of Pharmacology. Baylor College of Medicine, Houston.Texas 77030 ¡M. A. Z.. L C. Y./: and Vanderbill University School of Medicine. Departments of Medicine and Cell Biology. Nashville. Tennessee 37232 IR. J. CI

ABSTRACT

The DiFi colorectal carcinoma cell line, derived From a patient withfamilial adenomatous polvposis. was examined for gene expression andproduction of the autocrine growth factor transforming growth factor a(TGF-«) and for epidermal growth factor receptor (EGFR) gene expression and gene copy number. DiFi cells expressed TGF-a transcripts asidentified on Northern (RNA) blots. Addition of TGF-a ( 10 ng/ml) or EOF110 ng/ml) to DiFi cell cultures (lacking I <.1 or serum) up-regulated DiFicell basal T(iF-» mRNA levels, suggesting that autoinduction of TGF-a

occurs in these cells. DiFi cell cultures in log phase growth secreted measurable amounts of TGF-u (347 pg/10* cells/24 h) into their culture me

dium, as determined by radioimmunoassay. DiFi cells showed strong over-

expression of the EGFR gene on Northern blots relative to three othercolon cancer cell lines examined. Immunoperoxidase staining showed enhanced I I.I K expression in a cell subpopulation among the original (uncultured) ascitic fluid cells from which the DiFi cell line was established.This cell subpopulation was observed to expand dramatically betweenpassages 1 and 25. Immune complex kinase assay of DiFi cells showed thatEGFR were functional as determined by their ability to autophosphory-

late. The EGFR gene in these cells was not found to be rearranged orgenetically altered using Southern blot analysis. Dot blot analysis of DiFicell DNA revealed EGFR gene amplification in the range of 60-80 copies/cell, which is approximately twice the copy number seen in A-431 epider-

moid carcinoma cells. To our knowledge DiFi cells represent the firstexample of EGFR gene amplification in a colorectal adenocarcinoma.Because Ilili colorectal cancer cells uniquely show production and auto-induction of TGF-a in addition to amplification and overexpression of the

EGFR gene, these cells represent a valuable tool for studying the role(s) ofthe EGFR in the regulation of tumor cell growth.

INTRODUCTIONEOF' is a polypeptide growth factor produced by many normal

human tissues and is present in normal serum. TGF-a, a homologue

of EOF. is produced by a variety of malignant and normal cell types.Both TGF-a and EOF are known to bind to the same cell surface

receptor, the EGFR. and they produce a similar biological response(reviewed in Refs. I and 2).

Received 9/8/92; accepted 1/14/93.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 with18 U.S.C. Section 1734 solely to indicate this fact.

1Support for these studies was provided by Health Future Foundation Funds. Creigh-

ton University Department of Medicine Research Fund. The University of Texas M. D.Anderson Cancer Center New Program Funds, and NIH grant CA5039I (L. C. Y.).Preliminary data were presented at the proceedings of the Fourth International Symposiumon Colorectal Cancer. Kobe. Japan. November 1989.

2 To whom requests for reprints should be addressed, at Creighton Cancer Center.

Creighton University School of Medicine. 24th and California Street. Omaha. NE 68178.1The abbreviations used are: EGF. epidermal growth factor; TGF-a, transforming

growth factor-a; EGFR. epidermal growth factor receptor(s); RIA, radioimmunoassay:ATCC, American Type Culture Collection: FBS. fetal bovine serum; SSC. standardsodium citrate: 10F medium, McCoy's 5A medium (GIBCO) containing UK? FBS;SF-EGF medium. McCoy's 5A medium containing 4 ug/ml transferrin and 20 ug/ml

insulin but lacking serum and EGF: DMSO. dimethyl sulfoxide; TPA, 12-O-tetrade-canoylphorbol-l3-acetate; cDNA, complementary DNA.

Alterations in EGFR number or structure in a given cell mayinfluence the growth properties of that cell. For example, when NIH3T3 cells were transfected with a vector containing the full-length

EGFR gene (which induces EGFR overproduction), they became fullytransformed (3). Additionally, in subclones of A-431 cells having

different degrees of EGFR gene amplification and expression, thelevel of EGFR observed is reported to be proportional to the ability ofthe cells to grow both in vivo and in vitro (4). The oncogenic properties of \-erhB. which encodes a truncated version of the EGFR (5).

indicate that an altered EGFR can directly lead to tumorigenesis.Overproduction of EGFR has been reponed for squamous cell

carcinomas (lung, skin, oral cavity, and esophagus), brain tumors(glioblastomas and meningiomas). breast adenocarcinomas (90%),and colon adenocarcinomas (moderately well differentiated but notpoorly differentiated) (6-13). In some tumors, the overproduction of

EGFR is correlated with amplification of the number of copies of theEGFR gene (7. 10. II). In gliomas, the amplified EGFR gene isfrequently rearranged, suggesting that this gene mutation may beassociated with development of the tumor ( 10). Yet. in the majority ofcancers that overproduce EGFR, such overproduction appears to occur through mechanisms other than gene amplification. An analysis of101 cancers, including carcinomas, sarcomas, lymphomas, and leu-

kemias. showed that only one squamous cell carcinoma had amplification of the EGFR gene (14). Whereas some primary colonie adenocarcinomas and colon carcinoma cell lines showed enhanced EGFRexpression, none displayed gene amplification (12, 14, 15).

The initial purpose of our study was to determine EGFR gene copynumber and expression (mRNA) in the DiFi colorectal carcinoma cellline, which was established from a patient with familial adenomatouspolyposis (16). Because these studies revealed dramatic EGFR geneamplification/overexpression in DiFi cells ( 17), we conducted furtherstudies on cell surface EGFR expression, EGFR kinase activity.TGF-a production, and potential TGF-a autoinduction in these cells

to determine their value in understanding the role of the EGFR inhuman colon tumor cell growth.

MATERIALS AND METHODS

Cell Culture. DiFi cells and SW403 colon cancer cells (ATCC CCL 230)were routinely cultured (without udded EGF or TGF-a) in a 50:50 mixture ofL-15:Dulbecco's modified Eagle's culture medium (both from GIBCO. Grand

Island. NY) containing lO'/r FBS plus insulin (5 ug/ml; Sigma Chemical

Company, St. Louis, MO), transferrin (5 ug/ml; Sigma), and sodium selenite(5 ng/ml: Collaborative Research. Waltham. MA) and plated into standard75-cm- plastic tissue culture flasks or 850-cnr plastic roller bottles (Coming

Glass, Corning. NY). The three remaining SW lines. SW480. SW620 (ATCCCCL 228 and CCL 227). and SW742 (from Dr. Benjamin Drewinko, TheUniversity of Texas M. D. Anderson Cancer Center. Houston. TX), were grownin L-15 medium (modified) containing i-glutamine. supplemented with \07cFBS. insulin (5 ug/ml). and glutathione (16 ug/ml). A-431 cells (ATCC CRL

1555) used for EGFR gene amplification comparison with DiFi cells weremaintained in Dulhecco's modified Eagle's medium with 4.5 g/liter glucose

1630

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DiFi CELL TGF-u PRODUCTION AND AUTOINDUCTION

and 10% FBS. All cell cultures were incubated at 37°Cin a humidified

atmosphere of 95% air/5% CO2.Extraction of Nucleic Acids from Cultured Cells for Blot Analysis.

Approximately IO1*cells from each of the cell lines. DiFi, SW620, SW480,

SW403. SW742. and A-431. were pooled, collected by cemrifugation. and

subjected to a modified guanidinium isothiocyanate/cesium chloride method(18) for isolation of total cellular RNAand high relative molecular mass DNA.

Northern Blot Analysis. Polyadenylated RNA was selected from each cellline by chromatography of total RNA on oligo(dT)-cellulose columns (19).This mRNA (5 ug/lane) from each cell line was size fractionated by electro-

phoresis in a 1.1% agarose gel containing 2.2 Mformaldehyde and transferredto nylon filters using 20x SSC (IX SSC = 0.15 M sodium chloride. 0.015 M

sodium citrate).DNA Dot Blot Analysis. DNA of high relative molecular mass, which was

obtained from the guanidinium/CsCI interphase (above), was prepared fromeach cell line using a standard proteinase K and phenol/chloroform extractionprocedure. After each DNA sample was denatured (0.4 MNaOH). boiled, andneutralized (0.22 M Tris, pH 9.0, and l M HCl), 16 ug of each were seriallydiluted (2-fold at each step) with equal volumes of 10X SSC and subsequently

spotted onto nylon filters. Samples were then washed, air dried, and baked for2 h in a vacuum oven at 80°C.In addition. DNA was extracted for dot blot

analysis from normal human leukocytes (as described in Ref. 20) and from aliver metastasis (as for DiFi cells, above; frozen tissue supplied by Dr. Paul B.Lansing. New Orleans. LA) of the individual from whom DiFi cells were

derived.Hybridization. Filters were hybridized for 72 h at 42°Cwith 32P-DNA

probes which had been radiolabeled using the oligo primer extension method(21) to a specific activity of 1-3 x IO1*cpm/|jg of DNA. Specific cDNA

molecular probes used for TGF-a and chick ß-actingenes were described in

detail by Untawale and Blick (22). cDNA EGFR gene probes used in this studyincluded HER-A64-1 (a 1.84-kilobase EcoRl insert in pUC-1 encoding theEGFR external and transmembrane domains; 23). phEGFR-1 (an 880-base pairE(Y)RI insert in pBR322. containing sequence information for about 190 car-boxy-terminal amino acids of the EGFR plus a portion of the 3'-untranslated

region of the receptor mRNA; 24), pE7 (a 2.4-kilobase Clai insert encoding theEGFR external, transmembrane, and protein kinase domains; 25). and HER-A64-3 (a 768-base pair fragment encoding the EGFR protein kinase domain;

22).To study the effects of exogenous EGF & TGF-a on DiFi cell TGF-a gene

expression, an RNA probe for the human TGF-a gene (corresponding to thecDNA insert of plasmid pTGF-C 1) that was labeled with [32P]a-UTP was usedas previously described (26). The filters were subsequently washed at 60°Cfor

60 min using a solution of 0.1 X SSC and 0.1% sodium dodecyl sulfate, airdried, and autoradiographed using Kodak XRP-1 film with intensifying screensat -70°C. In DNA and RNA blots, either the chick ß-actingene probe or a

cDNA probe for the cyclophilin gene was used to reprobe filters as describedpreviously (22. 27) in order to verify that equal amounts of nucleic acid hadbeen loaded in each sample lane. Densitometry was performed for signalintensity on autoradiograms of DNA and RNA blots using a Hoefer scanningdensitometer (GS-3000).

Immunocytochemical Staining. Uncultured metastatic cells from asciticfluid aspirated from an abdominal cyst of the patient from whom DiFi cellswere derived, as well as cultured DiFi cells at passages 1 and 25. were testedfor the presence of EGF receptors by an immunoperoxidase procedure (28).Smears of the uncultured ascitic fluid cells and cytospins (prepared by standardprocedures) of the cultured DiFi cells were examined for reactivity with theanti-EGFR monoclonal antibody Rl (Amersham. Arlington Heights. IL). An

tibody reactivity was determined using the Vectastain ABC avidin/biotin reagent (for immunoperoxidase) to mouse IgG (Vector Laboratories. Burlin-game. CA). in which diaminobenzidine (l mg/ml in Tris-HCl. pH 7.2) was

used as substrate. The cells were counterstained with méthylènegreen.Adaptation of DiFi Cells to Growth in Chemically Defined Medium for

Conditioned Medium Studies. DiFi cells were adapted to 10F medium in ahumidified atmosphere of 94% air/6% CO2 (29, 30). Following 4 to 6 passagesin 10F medium, cells were changed to chemically defined medium, i.e., SF-EGF medium (31). Although this procedure produced an increase in the cell-doubling time from 19.5 h (10F medium) to 32.4 h (SF-EGF medium), cell

morphology and viability (>95%) were unchanged. DiFi cells have been

maintained under these conditions for at least 3 months (representing morethan 12 passages).

DiFi Cell-conditioned Medium. DiFi cells grown to either 50-80% or80-100% confluence were allowed to condition fresh SF-EGF medium (above)

for 72 h. Control flasks were treated identically except that they containedmedium but no cells. Media were dialyzed against phosphate-buffered saline

(GIBCO). which contained 0.1 mm phenylmethylsulfonyl fluoride and 0.025m.\i leupeptin. for 2 h at 4°C.followed by an overnight dialysis against waterand one additional change of water for 2 h at 4°C.The dialyzed. conditioned

medium was lyophilized and concentrated 10-fold by resuspending in 1/10volume of phosphate-buffered saline.

TGF-a Radioimmunoassay of DiFi Cell-conditioned Medium. TGF-aconcentrations were determined for cell-conditioned medium (and control flask

medium) using an RIA kit obtained from Biotope (Seattle, WA). All samplesand standards were pretreated with Biotope pretreatment buffer (10 ul/100 ulof sample) which consists of an ionic detergent and a reducing agent. Thesamples in pretreatment buffer were then heated to 90°Cfor 1 min. After

cooling. 50 ul of sample were tested in each reaction tube. Samples wereincubated overnight (>16 h) at 4°C.After separation of bound from free

radiolabeled TGF-a. samples were counted on a Beckman gamma counter(Beckman Instruments. Fullerton. CA). TGF-a concentrations were then de

termined from a standard curve prepared with each Biotope assay. The lowersensitivity limit of this RIA is 0.312 nv, (1.87 ng/ml) for TGF-a. It was

necessary to allow cells to condition the medium for 72 h in order to obtainTGF-a values falling within the linear range (0.312-10 nin) of the standard

curve for the assay. All values reponed were within this range. Ail assays wereperformed on duplicate samples (<5% coefficient of variation).

Effect of Exogenous EGF and TGF-a on TGF-a Gene Expression. DiFi

cells were removed from their routine culture medium and grown for 24 h inserum-free L-15 medium (GIBCO) lacking insulin, transferrin, selenium. EGF.or TGF-a. They were then grown for 2 h in fresh medium lacking EGF(control) or containing either 10 ng/ml EGF. 10 ng/ml TGF-a, 10 ng/ml

DMSO. or 10 ng/ml TPA. (As a second control. SW620 cells were treated inthe same manner as the DiFi cell control culture.) Total RNA was then extracted from the cell cultures and selected for polyadenylated RNA. which wasanalyzed by Northern blot as described above.

Immune Complex Kinase Assay. To determine whether DiFi EGFR werebiologically functional, their ability to become autophosphorylated was determined by an immune complex kinase assay (Fig. 6; 32). DiFi cells grown to logphase in their routine culture media were lysed in Ripa B buffer (10 ug/mlleupeptin. 20 mm Na2HPO4. 150 imi NaCl, 5 imi EDTA, 5 m.viphenylmethylsulfonyl fluoride. 1% aprotinin, pH 7.4) and clarified by centrifugation at20.000 x g for 10 min at 4°C.Cell lysate samples (100 Mg of protein/each

sample) were treated with the anti-EGFR monoclonal antibody Rl

(Amersham) to immunoprecipitate DiFi cell EGFR. Rl/EGFR complexes wereharvested, washed, and assayed for immune complex kinase activity as described before (33) and subsequently electrophoretically fractionated on 8%polyacrylamide gels (containing sodium dodecyl sulfate) and autoradiographedon Kodak X-Omat film at -70°C.

RESULTS

EGFR Gene Amplification in DiFi Cells. Southern (DNA) hy

bridization analysis of DiFi cell DNA using the pE7 probe showed the

presence of EGFR gene amplification (Fig. 1) relative to three other

colon cancer cell lines. SW480, SW403, and SW620. On Southern

blots, EcoRl and Hindlll digests of DiFi cell DNA hybridized with

pE7 produced banding patterns that were the same as those previously

reported for each restriction enzyme (25. 34). DNA dot blot analysis

was used to measure the degree of this EGFR gene amplification (Fig.2) using the HER-A64-3 probe (an £coRI subfragment of probe pE7);

each blot was reprobed for the chick ß-actin gene in order to dem

onstrate the relative amounts of DNA loaded in each lane (Fig. 2). In

Fig. 2A, DiFi cell EGFR gene amplification was determined (bydensitometry ) to be twice as high as that found in cell line A-431 (23),

indicating a 30- to 40-fold amplification of the gene in DiFi. Fig. 2B

shows that DiFi cells display this same level of EGFR gene amplifi

cation relative to DNA from three other human colon cancer cell lines

and from normal human colonie mucosa. Dot blot studies of DNA

from an hepatic metastasis of the rectal carcinoma in this patient alsorevealed a 23-fold EGFR gene amplification relative to the WBC

1631

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DiFi CELL TOF-o PRODUCTION AND AUTOINDUCTION

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Hind III EcoR1Fig. 1. Southern (DNA) hybridization using the EGFR cDNA probe pE7 with DNA

from DiFi. SW480. SW403. and SW620 colorectal carcinoma cell lines and from normalhuman WBCs. DNA samples {10 ug) were digested with the restriction enzymes HindUlor EíYíRIas indicated. The autoradiogram shown was exposed to optimally resolve theDiFi banding pattern; however, this exposure time is suboptimal for demonstrating thehybridization signal in DNA samples lacking EGFR gene amplification. Kb, kilobases.

DNA of the patient, as determined by densitometry (blot not shown).DNA blot analysis of DiFi cell DNA using the phEGFR-l probe(encoding the 3' end of the gene) and the HER-A64-1 probe (encoding the 5' end of the gene) showed EGFR gene amplification (data not

shown), indicating that the entire gene is amplified in these cells. Thelevel of EGFR gene amplification reported here has been maintainedby DiFi cells with repeated subculturing (>100 passages).

EGFR and TGF-a Transcripts. Northern (RNA) hybridizationstudies showed that transcripts for TGF-a were clearly expressed in

DiFi cells (Fig. 3) under routine culture conditions. Under such conditions, DiFi cells showed markedly elevated EGFR gene mRNAlevels on Northern blots (Fig. 4) relative to the expression seen in thecolon cancer cell lines SW403, SW480, and SW620. The EGFR genemRNA species observed (10.5, 6.1, and 5.8 kilobases; Fig. 4) areconsistent with those detected in normal placenta (23). In addition tothese major mRNA bands, two fainter bands (2.8 and 1.5 kilobases)were observed on DiFi cell Northern blots after long autoradiographicexposure times (data not shown). These bands are thought to representlow-abundance variant transcripts of the EGFR gene or possibly tran

scripts of other genes that have structural similarity to the EGFR gene(23).

Detection of Cell Surface EGFR on DiFi Cells. Analysis byimmunoperoxida.se staining showed what appeared to be stronglyenhanced expression of EGFR in a subpopulation of cells from cyto-

logical preparations of ascitic fluid (Fig. 5A ) from the patient who wasthe source of DiFi cells. This population of cells with high EGFRexpression was observed to expand as the DiFi cell culture (initiatedfrom this same ascitic fluid sample) progressed from the first passageto the 25th passage (Fig. 5, B and C).

Autophosphorylation Ability of DiFi EGFR. The immune complex kinase assay shows a 36-fold increase in EGFR autophosphory-

lation specific activity relative to that of another colon cancer cell line,SW620, as determined by densitometry (Fig. 6). The lower molecularweight bands in the SW620 lane remain to be identified but mayrepresent kinase activity of a truncated EGFR.

TGF-a Production by DiFi Cells. DiFi cells at different confluence levels secreted measurable amounts of TGF-a into SF-EGF

medium, i.e., the difference between the group mean values determined by RIA (control versus 50-80% confluent) is significant(P = 0.0245). DiFi cells at 50-80% confluence were found to produce347 pg/106 cells/24 h of TGF-a (corrected for control, = average of

5 determinations). This is probably the most informative value because it represents cells in the log phase of growth. In contrast, a lowervalue of 86 pg/106 cells/24 h (corrected for control. = average of 2

determinations) was obtained from cultures that were 80-100% confluent, probably representing a shift in TGF-a secretion reflecting the

decreased protein synthesis and growth arrest generally observed inconfluent cultures.

A determination of the cell-associated TGF-a fraction by themethod of Zorbas and Yeoman (35) showed the level of acid-extract-able TGF-a to be 140 pg/106 cells after 24 h in SF-EGF medium. This

amount of acid-extractable TGF-a was estimated to be about 30% ofthe total TGF-a production (secreted plus cell associated) using theconditioned medium value for soluble TGF-a reported above (347pg/106 cells/24 h).

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Fig. 2. DNA dot blot analysis for EGFR genes in DNA extracted from: A, normalWBCs from the patient who was the source of DiFi cells, DiFi cells, A-431 epidermoidcarcinoma cells; B, normal colonie mucosa (N/) from an unrelated patient, and thecolorectal cancer cell lines DiFi. SW480, SW620, and SW742. The cDNA probe HER-A64-3 was used for hybridization blots of serially diluted DNA samples as described in"Materials and Methods." The blot was rehybridized with a chick ß-actingene probe to

verify equal DNA sample loading.

1632

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DiFi CELL TOF-(i PRODUCTION AND AlTOINDl CTION

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EGFR transcripts, autophosphorylation, and cell surface staining.Southern (DNA) hybridization studies did not indicate that the EGFRgene in DiFi cells is rearranged or genetically altered using either thepE7 or phEGFR-1 cDNA probes, which together span approximately

81% of the EGFR gene. Moreover, Northern analysis of DiFi cellmRNA showed normal-sized EGFR transcripts (23). The receptor

appears to be biologically functional, as indicated by its ability toautophosphorylate.

Immunocytochemical data also indicate that this EGFR gene amplification already existed in primary metastatic cells from the patientwho was the source of DiFi cells. We further established this point byDNA blot analysis of a liver metastasis removed from this individual.Tissue culture conditions used to establish the DiFi cell line (16)apparently selected for a subpopulation of cells having elevated EGFRexpression as the number of passages increased. Selection of theEGFR-overexpressing subpopulation of cells from the primary rectal

tumor of the patient may have occurred during metastatic spread to theliver because increased EGFR expression has been correlated withenhanced growth ability at metastatic sites in colorectal cancer progression (36).

Some sporadic colon adenocarcinomas show enhanced EGFR expression without gene amplification (12, 15). Our observation ofEGFR overexpression coupled with EGFR gene amplification in DiFirectal adenocarcinoma cells is interesting because, although EGFRgene amplification has been reported in some squamous cell carcinomas (7, 14) and brain tumors (10), the only adenocarcinomas previously reported to show amplification of this gene were breast cancers(11).

Comparison of structure and function of EGFR in DiFi cells andA-431 vulval epidermoid carcinoma cells shows several notable dif-

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Fig. 3. Northern blot analysis demonstrating the expression of the TGF-a gene (4.8kilobases (kb)]. Lanes contain 5 ng of mRNA isolated from DiFi cells, normal humancolonie mucosa (N/), and a sporadic primary human colon carcinoma (T). 28S and 18S,positions of human rRNA species. The blot was rehybridized with the chick ß-actingeneprobe to verify equal mRNA sample loading.

Effects of Exogenous TGF-a and EGF on TGF-a Gene Expression. As shown in Fig. 7, exogenous EGF ( 10 ng/ml ) and TGF-a ( 10ng/ml) both induce an up-regulation of TGF-a gene expression inDiFi cells (1.7- and 2.9-fold, respectively, determined densitometri-cally). TPA (10 ng/ml) also increases the level of TGF-a message inthese cells (2.15-fold), whereas DMSO (10 ng/ml) does not, as deter

mined by densitometry. Untreated SW620 cells produce about 30%more TGF-a transcripts (densitometrically determined) than the DiFi

cell control (cultured without growth factors or FBS, Fig. 7). butSW620 cells do not display TGF-a autoinduction in response toexogenous EGF or TGF-a (R. Coffey, unpublished observations).

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DISCUSSION

DiFi appears to represent a unique colorectal cancer cell line thatdisplays both amplification of the EGFR gene and autoinduction ofTGF-a by exogenous EGF and TGF-a. Assay of DiFi cell DNA

showed that the degree of EGFR gene amplification seen in these cellscorrelates with the observed EGFR overexpression level in terms of

Fig. 4. Northern blot analysis demonstrating the expression of the EGFR gene usingcDNA probe pE7. Lanes contain 5 ug of mRNA isolated from colorectal cancer cell linesDiFi. SW403. SW480. and SW620. The autoradiogram shown was exposed to optimallyresolve the DiFi cell transcripts; however, this exposure time is suboptimal for demonstrating the hybridization signal in RNA samples lacking EGFR overexpression. EqualmRNA sample loading was verified as described in "Materials and Methods." Kb. kilo-

bases.

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DiFi CELL TGF-a PRODUCTION AND AUTOINDUCTION

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UP*- ' '-'f ... »=

Fig. 5. Immunocytochemical staining of DiFi cells using anti-EGFR antibody. Cytological preparations of uncultured ascitic fluid (A I and cultured DiFi cells at passage 1 (B) orpassage 25 (C) were examined using a monoclonal anti-EGFR antibody. Arrm-s, some of the cells staining positively for EGFR.

U_

Q

EGFR

number of EGFR in DiFi cells (4.8 X IO6 receptors/cell; 32) is 2- to4-fold greater than is found in A-431 cells (1-3 X IO6 receptors/cell,

39). Nevertheless, both DiFi and A-431 cell cultures have a similar

growth response to the addition of exogenous EOF in that high EOFdoses (concentrations >1 nsi EOF) produce growth inhibition,whereas lower EOF concentrations induce a mitogenic effect (39, 40).Thus, DiFi cells will be useful in studying the role of EGFR in tumorcell growth regulation.

Our data suggest that production of TGF-a and overproduction of

EGFR may provide a selective growth advantage in DiFi cells. The

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Fig. 6. Immune complex kinase assay of EGFR from colorectal cancer cell lines DiFiand SW620 showing degree of receptor autophosphorylation. Arrow, position of the EGFRhand on autoradiographs of 8% polyacrylamide gels.

ferences. The level of EGFR gene amplification is twice as high inDiFi cells as in A-431 cells. A-431 cells are known to show a rear

ranged EGFR gene (23) which generates a truncated transcript (2.8kilobase) encoding only the extracellular EGF-binding domain. Theamplified EGFR gene in A-431 cells has been reponed to be chro-

mosomally located (25, 37). In DiFi cells in situ hybridization usinga biotinylated cDNA EGFR gene probe has recently revealed theamplified EGFR genes to be present in extrachromosomal closedcircular episomes (38). Competitive binding studies show that the

•¿�*•Cyclophilin

Fig. 7. EOF and TGF-a induction of TGF-a mRNA in DiFi cells. The effect of EGF(10 ng/ml). TGF-a (10 ng/ml). DMSO (10 ng/ml), and TPA (10 ng/ml) on the expressionof TGF-a transcripts was determined by Northern blot analysis. The level of TGF-amRNA in SW620 cells is shown for comparison. The control represents the level ofTGF-a message in DiFi cells grown in serum-free, unsupplemented medium lackingexogenously added factors. The blot was rehybridized with a cyclophilin gene probe toverify equal RNA sample loading.

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D¡F¡CEU. TGF-a PRODUCTION AND AUTO1NDUCT1ON

fact that DiFi cells secrete TGF-a and have detectable cell-associatedTGF-a (proTGF-a; 41) indicates that they may gain this growth ?advantage via an autocrine-related mechanism. proTGF-a has been

shown to play a role in EGFR transmembrane signaling in anothercolon carcinoma cell line, HCT1 16 (35). Thus, DiFi cells may possessa similar proTGF-a/EGFR-mediated mechanism involving the bind- 9.ing of cell surface-associated proTGF-a to (overexpressed) EGFR on

adjacent cells. The observation that DiFi cells are able to proliferate in |0medium free of serum or EGF suggests that the amount of endogenousTGF-a that they produce is sufficient to stimulate growth under theseconditions. Autoinduction of TGF-a expression by endogenousTGF-a production may also function to potentiate growth in DiFicells. 12'

Our observations of DiFi cell up-regulation of TGF-a gene expression in response to exogenously added EGF, TGF-a, and TPA are I-1-

similar to those reported for cultured human keratinocytes (26, 42).DiFi cells will be valuable in our ongoing studies of the cellular u.mechanisms involved in regulating TGF-a autoinduction in normal

versus transformed cells (43). To date, the only other known example |5of TGF-a autoinduction in a colon cancer cell line is LIM 1215 cells,

derived from a patient with the hereditary nonpolyposis colorectalcancer syndrome (43). Thus, our results suggest the need to investigate a possible role for TGF-a/EGFR autocrine mechanisms in the

development of hereditary colorectal cancers.The production of TGF-a and presence of EGFR gene amplifica

tion in DiFi cells represents a unique opportunity to study the role ofEGFR expression in tumor growth. This has led to our ongoing studiesof the growth effects of anti-EGFR antibodies (44) and an EGFR-

specific tyrosine kinase inhibitor (40) on DiFi cells. In addition, thesecells should be useful in studying cellular responses to EGFR-anti-

sense oligonucleotides and to synthetic EGF peptide analogues (45).The role of other EGFR ligands, such as amphiregulin and cripto (46),in cell growth control could also be examined using DiFi cells. Moreover. the high EGFR expression in DiFi cells could be valuable instudying EGFR-binding proteins by immunoprecipitation analysis.

ACKNOWLEDGMENTS

The results reported in this paper include research performed by one of theauthors (S. U.) as part of the requirements for a master's thesis. We thank Dr.

Asit Chakraborty of Creighton Cancer Center, Omaha. NE. for assistance in theanalysis of colon carcinoma cell lines by Southern and Northern blot. We aregrateful to Melanie Chun of Creighton Cancer Center. Omaha. NE. and DebraM. Lytle of the University of Nebraska Medical Center. Omaha, NE. forassistance with densitometric scans of Southern and Northern blots. We thankthe referring physician. Dr. Paul B. Lansing of New Orleans, LA. for providinga tissue sample from the patient who was the source of DiFi cells. The use ofhuman materials in establishing the DiFi colorectal carcinoma cell line was infull compliance with the guidelines and approval of the appropriate reviewboards for human experimentation.

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1993;53:1630-1636. Cancer Res   Seema Untawale, Mark A. Zorbas, Clague P. Hodgson, et al.   Epidermal Growth Factor Receptor Genea Colorectal Carcinoma Cell Line (DiFi) with an Amplified

Production and Autoinduction inαTransforming Growth Factor-

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