In vitro transformation of rat esophageal epithelial cells with N-nitrosobenzylmethylamine

Using an explant/cell culture system, rat esophageal epithelial cells were transformed in vitro by exposure to N-nitroso-N-benzyl-N-methylamine (BMNA). Twelve esophageal explant cultures per group were exposed twice (at days 1 and 7) to 0.0, 2.5, 5.0 or 10.0 μg BMNA/ml of medium. After incubation for 60–90 days, epithelial cells in primary cultures treated with all three concentrations of BMNA could be subcultured and cell lines were developed. The number of primary cultures and the number of subsequently developed epithelial cell lines was carcinogen-dose-dependent. Cell lines could only be established from carcinogen treated explants. Electron microscopy revealed that the BMNA-treated cell lines contained morphological markers of esophageal epithelial cells; i.e., numerous tonofilaments and junctional complexes, even after prolonged subculture. By immunofluorescence, the cells reacted positively with antibodies prepared to mouse skin prekeratins (K1 and K2). Two cell lines (from the 5 μg BMNA/ml group) were able to grow in soft agar and produce palpable tumors upon injection into syngeneic recipients. These tumors possessed the histological features of squamous cell carcinomas.