Identification of early carcinogen-induced changes in nutritional and substrate requirements in cultured tracheal...

The purpose of our studies was to determine whether specific differences in nutritional and/or substrate requirements exist between normal and carcinogen treated tracheal epithelial cells. Epithelial cells were collected from control tracheas or from tracheas exposed in vivo for 4 weeks to the carcinogen 7,12-dimethylbenz[a]anthracene (DMBA). This carcinogen exposure was shown in previous studies (18) to induce various types of transformed epithelial cell phenotypes. The cells were cultured under 7 different culture conditions, one of which was designed to provide optimum growth conditions for all cells (nonselective condition = SC0) and contained F12, conditioning factors from 3T3 fibroblasts, Dulbecco's minimal essential medium (DME), insulin, transferrin, hydrocortisone, fetal bovine serum (FBS) and bovine hypothalamus extract. By removing one or more factors from SC0 we hoped to design conditions selecting against normal and for "carcinogen altered" cells. It was found that normal cells require collagen and conditioning factors (CF) (produced from 3T3 fibroblasts grown in DME + 2% FBS) for growth in hormone supplemented medium. Increasing the serum concentration to 10% did not compensate for the collagen and CF requirements. In comparison, cells from carcinogen-exposed tracheas clearly had reduced nutritional and substrate requirements. Such cells grew for at least 30 days without collagen substratum as long as CF were present or without CF as long as collagen was present. High serum concentration replaced the requirement for both collagen and CF. Immortalization and anchorage independence of growth occurred in all cultures derived from DMBA-exposed tracheas except in those maintained in high serum but without DME, collagen and CF. These studies indicate that "carcinogen-altered" cells can be identified and selected for based on substrate and nutrient requirements. This should be useful in designing a quantitative epithelial transformation system.