Untawale S, Zorbas M A, Hodgson C P, Coffey R J, Gallick G E, North S M, Wildrick D M, Olive M, Blick M, Yeoman L C
Creighton Cancer Center, Creighton University School of Medicine, Omaha, Nebraska 68178.
Cancer Res. 1993 Apr 1;53(7):1630-6.
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.
对源自一名家族性腺瘤性息肉病患者的DiFi结肠癌细胞系进行了检测，分析其自分泌生长因子转化生长因子α（TGF-α）的基因表达和产生情况，以及表皮生长因子受体（EGFR）的基因表达和基因拷贝数。通过Northern（RNA）印迹法鉴定，DiFi细胞表达TGF-α转录本。向DiFi细胞培养物（不含表皮生长因子或血清）中添加TGF-α（10 ng/ml）或表皮生长因子（EGF，10 ng/ml）会上调DiFi细胞基础TGF-α mRNA水平，这表明这些细胞中存在TGF-α的自诱导现象。通过放射免疫测定法确定，处于对数期生长的DiFi细胞培养物向其培养基中分泌了可测量量的TGF-α（347 pg/10⁶细胞/24小时）。与所检测的其他三种结肠癌细胞系相比，DiFi细胞在Northern印迹上显示出EGFR基因的强烈过表达。免疫过氧化物酶染色显示，在建立DiFi细胞系的原始（未培养的）腹水细胞中的一个细胞亚群中，EGFR表达增强。观察到该细胞亚群在第1代至第25代之间显著扩增。对DiFi细胞进行免疫复合物激酶分析表明，EGFR具有自磷酸化能力，从而证明其具有功能。使用Southern印迹分析未发现这些细胞中的EGFR基因发生重排或基因改变。对DiFi细胞DNA进行斑点印迹分析显示，EGFR基因扩增范围为60 - 80拷贝/细胞，约为A - 431表皮样癌细胞中观察到的拷贝数的两倍。据我们所知，DiFi细胞是结肠腺癌中EGFR基因扩增的首个实例。由于DiFi结肠癌细胞除了EGFR基因的扩增和过表达外，还独特地表现出TGF-α的产生和自诱导，因此这些细胞是研究EGFR在肿瘤细胞生长调节中的作用的宝贵工具。
