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 presentstudy, in vitro transformation of spontaneously immortal hamsterpancreatic duct cells following exposure to 20 mM NNK for 1,3,5and 7 days is described. NNK imparted a dose-dependent and time-dependenttoxicity to pancreatic duct cells in vitro. After NNK treatment,duct cells were grown either in complete duct medium (CDM) orin the absence of bovine pituitary extract, epidermal growthfactor and Nu-seruin (incomplete duct medium, 1DM). Additionof NNK to the culture for 1 and 3 days did not affect the growthof the cells, whereas exposure of the cells for 5 and 7 dayswas inhibitory. One and 3 day NNK-treated cells were able togrow in the absence of growth factors and serum immediatelyafter the treatment without any inhibition of growth. Untreatedcells grew as a monolayer consisting of tightly packed polygonalcells with single nuclei. NNK treated cells also grew as a monolayerwith numerous mitotic figures and multi-nucleated large cells.The doubling time between the untreated (16 h) and NNK-treatedcells (14 h) was not significantly different prior to injectioninto the nude mice. NNK treated cells grown in 1DM displayedanchorage independency in soft-agar. The tumorigenicity of theuntreated and NNK treated cells (5x10⁶) was determined in nudemice. One and 3 day NNK-treated cells grown in CDM producedwell-differentiated, mucinous tumors with a lower frequency(2/4 sites) and longer duration, but produced tumors at a higherfrequency (4/4 sites) and shorter duration when grown in IDM.Five and 7 day NNK-treated cells grown in CDM did not produceany tumors; however, they produced tumors when grown in CDMfollowed by IDM (5/8 and 6/8 sites) with a shorter durationin 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-rasoncogene in tumor cells of 1 and 3 day NNK treatment. No mutationwas detected in tumor cells from 5 and 7 day treatment.