Transformation of hamster pancreatic duct cells by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), in vitro

The tobacco specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a potent carcinogen in laboratory animals. In the present study, in vitro transformation of spontaneously immortal hamster pancreatic duct cells following exposure to 20 mM NNK for 1,3,5 and 7 days is described. NNK imparted a dose-dependent and time-dependent toxicity to pancreatic duct cells in vitro. After NNK treatment, duct cells were grown either in complete duct medium (CDM) or in the absence of bovine pituitary extract, epidermal growth factor and Nu-seruin (incomplete duct medium, 1DM). Addition of NNK to the culture for 1 and 3 days did not affect the growth of the cells, whereas exposure of the cells for 5 and 7 days was inhibitory. One and 3 day NNK-treated cells were able to grow in the absence of growth factors and serum immediately after the treatment without any inhibition of growth. Untreated cells grew as a monolayer consisting of tightly packed polygonal cells with single nuclei. NNK treated cells also grew as a monolayer with numerous mitotic figures and multi-nucleated large cells. The doubling time between the untreated (16 h) and NNK-treated cells (14 h) was not significantly different prior to injection into the nude mice. NNK treated cells grown in 1DM displayed anchorage independency in soft-agar. The tumorigenicity of the untreated and NNK treated cells (5×106) was determined in nude mice. One and 3 day NNK-treated cells grown in CDM produced well-differentiated, mucinous tumors with a lower frequency (2/4 sites) and longer duration, but produced tumors at a higher frequency (4/4 sites) and shorter duration when grown in IDM. Five and 7 day NNK-treated cells grown in CDM did not produce any tumors; however, they produced tumors when grown in CDM followed by IDM (5/8 and 6/8 sites) with a shorter duration in nude mice. Analysis of DNA for k-ras mutation at codons 12, 13 and 61 showed G–A transition at codon 12 of the k-ras oncogene in tumor cells of 1 and 3 day NNK treatment. No mutation was detected in tumor cells from 5 and 7 day treatment.