Selection of carcinogen-altered rat tracheal epithelial cells preexposed to 7,12-dimethylbenz[a]anthracene by their loss of a need for pyruvate to survive in culture

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Abstract
Normal rat tracheal epithelial cells require exogenous pyruvate to survive in culture while carcinogen-altered cells do not. The aims of our study were, (i) to utilize this loss of requirement for pyruvate by carcinogen-altered cells as a way of selecting out and quantitating the numbers of altered cell populations induced in tracheas exposed to 200 μg 7,12-dimethylbenz[a]-anthracene (DMBA)-beeswax pellets for different lengths of time, (ii) to determine the constancy of these numbers at increasing time intervals after the exposure, (iii) to look for distinctive growth properties in cell populations that may be related to the exposure conditions. Tracheal implants were exposed for 2 weeks, 4 weeks or continuously to the 200 μg DMBA. At 2,6 and 9 months after the start of exposure, the tracheas were cut into pieces and primary cultures established from outgrowths in an enriched Waymouth's medium. After 2 weeks, pyruvate and insulin were removed from the medium for 2 weeks to kill the normal cells and leave the altered, i.e., the selected cell populations (SPC). The pyruvate and insulin were then returned to the medium to stimulate rapid expansion of the SPC. The length of DMBA exposure had a marked effect on the number of SPC induced in the tracheas. There were 1.8 SPC/trachea 2 months after the 2 weeks exposure, while 5.0 and 7.2 SPC were obtained from the 4 weeks and continuously exposed tracheas, respectively, at this time. The numbers of SPC did not increase with time after exposure indicating that the maximal number of initiation sites was fixed. Between 2 and 9 months the number of subculturable SPC increased from 67 to 88% in the 2 week exposed group, while 93–100% of the SPC were subculturable after the longer DMBA exposures. Only the SPC from the 2 week DMBA - 2 month group were distinguished by smaller numbers of cells surviving the selection medium and by a slower average growth rate compared to all the other experimental conditions.