Regulation of transformation frequency by exogenous and endogenous growth factors in rat tracheal epithelial cells

The purpose of our studies was to re-evaluate the rat tracheal epithelial (RTE) transformation system and to identify critical variables that affect the development of enhanced growth variants (EGV). The enhanced growth variant colony, which is a preneoplastic cell variant, is the quantifiable transformation endpoint in RTE cultures. Using a standard protocol the frequency of EGV colony formation was shown to be inversely related to the number of clonogenic cells (CFU) seeded per dish in control cultures as well as in cultures treated with the transforming agent 6-nitrochrysene (6-NC). Experiments showed that the major mechanisms that underlie the CFU density-dependent inhibition of EGV colony formation are depletion of growth factors from and accumulation of autocrine TGF-beta in the media. Thus the cells themselves are creating the selection environment, which allows only the EGVs to survive. The effects of agents such as 6-NC, which increase the frequency of EGV colony formation, are to induce a cellular phenotype that is less susceptible to the selection environment. We showed that TGF-beta-neutralizing antibodies added to the selection media significantly increased EGV colony formation in control cultures but not in 6-NC-exposed cultures. In addition we demonstrated that the development of EGV colonies is much less susceptible to inhibition by (exogenous) TGF-beta in 6-NC-exposed than in control cultures. Thus spontaneous and 6-NC EGV colony formation are distinguishable based on TGF-beta sensitivity. To conduct quantitative cell transformation experiments with RTE cells it is essential that the number of surviving CFU per dish is the same in control and treated cultures. Under the conditions used in the studies described here, 350-500 CFU per culture was found to be the optimum CFU density. Besides 6-NC, agents that have been shown to increase EGV colony frequency under conditions similar to those described here are nitrosamines, NNK, nickel compounds and X-rays.