Predisposition of cloned fetal hamster lung epithelial cells to transformation by a precarcinogen, benzo(a)pyrene, using growth hormone supplementation and collagen gel substratum.

A cloned fetal Syrian hamster lung epithelial cell line (M3E3/C3) was used to compare the influence of two different culture conditions on the degree of cellular differentiation and susceptibility of the cells to undergo malignant transformation by a precarcinogen, benzo(a)pyrene. Conventional conditions consisted of growth medium containing Roswell Park Memorial Institute Medium 1640, pyruvate, and fetal bovine serum and a substratum of plastic. Complex conditions comprised the growth medium supplemented with insulin, hydrocortisone, estradiol, epidermal growth factor, transferrin, and cholera toxin and a substratum of collagen gel. Under the complex culture conditions, there was extensive development of endoplasmic reticulum and Golgi vesicles, whereas under conventional conditions these organelles were only minimally developed. This was correlated with 1.5-1.8 times enhancement of ethoxycoumarin deethylase and reduced nicotinamide adenine dinucleotide phosphate-dependent cytochrome c reductase activities. Decomposition of added benz(a)anthracene into water-soluble compounds increased with the period of incubation and reached about 40% of initial benz(a)anthracene (50 micrograms/10 ml/flask) at 48 h under the complex conditions, whereas under the conventional conditions only less than 4% decomposition occurred. Benzo(a)pyrene in the dose range 2-8 micrograms/ml was strongly cytotoxic and caused significant anchorage independent transformation only under complex culture conditions. Transformed cells produced tumors in two of four hamsters during 8 months following s.c. injection within 48 h of birth. These results suggest that the complex culture conditions predisposed the cloned fetal epithelial cells to malignant transformation by benzo(a)pyrene through stimulation of cellular differentiation and development of enzyme systems capable of activating it metabolically.