title: Transforming growth factor-alpha production and autoinduction in a colorectal carcinoma cell line (DiFi) with an amplified epidermal growth factor receptor gene.
abstract: The DiFi colorectal carcinoma cell line, derived from a patient with familial adenomatous polyposis, was examined for gene expression and production of the autocrine growth factor transforming growth factor alpha (TGF-alpha) and for epidermal growth factor receptor (EGFR) gene expression and gene copy number. DiFi cells expressed TGF-alpha transcripts as identified on Northern (RNA) blots. Addition of TGF-alpha (10 ng/ml) or EGF (10 ng/ml) to DiFi cell cultures (lacking EGF or serum) up-regulated DiFi cell basal TGF-alpha mRNA levels, suggesting that autoinduction of TGF-alpha occurs in these cells. DiFi cell cultures in log phase growth secreted measurable amounts of TGF-alpha (347 pg/10(6) cells/24 h) into their culture medium, as determined by radioimmunoassay. DiFi cells showed strong overexpression of the EGFR gene on Northern blots relative to three other colon cancer cell lines examined. Immunoperoxidase staining showed enhanced EGFR 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 between passages 1 and 25. Immune complex kinase assay of DiFi cells showed that EGFR were functional as determined by their ability to autophosphorylate. 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 epidermoid carcinoma cells. To our knowledge DiFi cells represent the first example of EGFR gene amplification in a colorectal adenocarcinoma. Because DiFi colorectal cancer cells uniquely show production and auto-induction of TGF-alpha 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.
authors.affiliation: Creighton Cancer Center, Creighton University School of Medicine, Omaha, Nebraska 68178.
authors.forename: S
authors.initials: S
authors.lastname: Untawale
authors.forename: M A
authors.initials: MA
authors.lastname: Zorbas
authors.forename: C P
authors.initials: CP
authors.lastname: Hodgson
authors.forename: R J
authors.initials: RJ
authors.lastname: Coffey
authors.forename: G E
authors.initials: GE
authors.lastname: Gallick
authors.forename: S M
authors.initials: SM
authors.lastname: North
authors.forename: D M
authors.initials: DM
authors.lastname: Wildrick
authors.forename: M
authors.initials: M
authors.lastname: Olive
authors.forename: M
authors.initials: M
authors.lastname: Blick
authors.forename: L C
authors.initials: LC
authors.lastname: Yeoman
mesh_terms: D015152:Blotting, Northern; D015139:Blotting, Southern; D015179:Colorectal Neoplasms / Q000378:metabolism*; D004273:DNA, Neoplasm / Q000032:analysis*; D066246:ErbB Receptors / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D005784:Gene Amplification / Q000235:genetics*; D006801:Humans; D010766:Phosphorylation; D016211:Transforming Growth Factor alpha / Q000096:biosynthesis*; D014407:Tumor Cells, Cultured; D015854:Up-Regulation
chemical_list: D004273:DNA, Neoplasm; D016211:Transforming Growth Factor alpha; D066246:ErbB Receptors