Inhibitor production by normal rat tracheal epithelial cells influences the frequency of spontaneous and X-ray-induced enhanced growth variants.

A cell culture model was used to assay for the induction of cell populations with enhanced growth capacity in culture in irradiated normal rat tracheal epithelial cells (NTEC). When cultures were maintained in minimally enriched Ham's F-12 plus 5% fetal bovine serum, it was noted that increasing the seeding density from 1 to 100 colony forming units per 60 mm dish decreased the frequency of proliferating epithelial foci (PEF) scored 5-6 weeks after seeding, from 2.5 to 0.07% in controls and from 5 to 0.9% in irradiated cultures. When low density (50 colonies per dish) control cultures were fed medium conditioned by higher density control cultures (300 colonies per dish) the observed frequency of PEF was 10-fold lower than that observed in cultures fed fresh medium or cultures fed medium conditioned by a carcinogen-induced tracheal cell line. These data suggest that some factor(s) present in conditioned medium derived from higher density normal cell cultures inhibits the emergence of many PEF. If cultures are maintained in serum-free enriched Ham's, many (17-21%) PEF were observed in both control and irradiated cultures. Medium harvested from high density cultures maintained in minimally enriched Ham's F-12 (5% FBS) was found to maximally inhibit normal cell thymidine uptake and growth as well as induce cornified envelope formation (terminal differentiation) in proliferating normal cell cultures. Medium harvested from normal cultures maintained in serum-free enriched Ham's was not inhibitory to normal cells and did not induce cornified envelope formation. Subcultured PEF-derived populations and PEF in 4 to 5-week primary cultures also did not produce inhibitor(s). In addition, subcultured PEF-derived cells were not induced to form cornified envelopes in the presence of inhibitory conditioned medium. The data suggest that the elaboration of an inhibitor(s) by normal epithelial cells affects the emergence of PEF. Irradiated cells appear to have some growth advantage over spontaneous PEF in that they are more likely to survive and form PEF under growth conditions associated with higher levels of inhibitor(s) in the medium.