[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 January 20, 2022. © 1993 American Association for Cancer cancerres.aacrjournals.org Downloaded from