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 epithel ial (RTE) transformation system and to identify critical variables tha t affect the development of enhanced growth variants (EGV). The enhanc ed growth variant colony, which is a preneoplastic cell variant, is th e quantifiable transformation endpoint in RTE cultures. Using a standa rd protocol the frequency of EGV colony formation was shown to be inve rsely related to the number of clonogenic cells (CFU) seeded per dish in control cultures as well as in cultures treated with the transformi ng agent 6-nitrochrysene (6-NC). Experiments showed that the major mec hanisms that underlie the CFU density-dependent inhibition of EGV colo ny formation are depletion of growth factors from and accumulation of autocrine TGF-beta in the media. Thus the cells themselves are creatin g the selection environment, which allows only the EGVs to survive. Th e effects of agents such as 6-NC, which increase the frequency of EGV colony formation, are to induce a cellular phenotype that is less susc eptible to the selection environment. We showed that TGF-beta-neutrali zing antibodies added to the selection media significantly increased E GV colony formation in control cultures but not in 6-NC-exposed cultur es. In addition we demonstrated that the development of EGV colonies i s 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 condu ct quantitative cell transformation experiments with RTE cells it is e ssential that the number of surviving CFU per dish is the same in cont rol and treated cultures. Under the conditions used in the studies des cribed here, 350-500 CFU per culture was found to be the optimum CFU d ensity. Besides 6-NC, agents that have been shown to increase EGV colo ny frequency under conditions similar to those described here are nitr osamines, NNK, nickel compounds and X-rays.