Malignant transformation of human bronchial epithelial cells with the tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone

Abstract

Sufficient evidence has demonstrated that cigarette smoking is causally associated with various types of human cancers. In the United States, about 90% of deaths from lung cancer among men and 79% of those among women are associated with smoking. Tobacco-specific nitrosamines are formed from nicotine and related tobacco alkaloids and are the most carcinogenic compounds of tobacco smoke. The most potent N-nitrosamine contained in tobacco smoke is 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, NNK. In the our study, the oncogenic transforming effects of graded doses of NNK were examined using papillomavirus-immortalized human bronchial epithelial cells. Growth kinetics, saturation density, resistance to serum-induced terminal differentiation, anchorage independent growth and tumorigenicity in nude mice were used to investigate the various stages of transformation in bronchial epithelial cells. We show here that immortalized human bronchial epithelial cells in culture can be malignantly transformed by treatment with NNK (100 microg/ml or 400 microg/ml) for 7 days. Transformed cells produced progressively growing subcutaneous tumors upon inoculation into nude mice. Immunofluorescence staining for keratin expression confirmed the epithelial nature of the tumor cells. Increased expression of p16, beta-catenin and PCNA in the established cell lines were detected by immunofluorescence staining and quantified by confocal microscopy. These data suggested that NNK can induce malignant transformation of human bronchial epithelial cells, and the tumor cell lines established are useful models in investigating the carcinogenic mechanism(s) of NNK.