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Effects of Serum Concentration on the Expression of Carcinogen-induced Transformation in the C3H/10T CL8 Cell Line¹

Department of Experimental Therapeutics, Grace Cancer Drug Center, Roswell Park Memorial Institute, Buffalo, New York 14263

The C3H/10T CL8 cell line (10T) is being widely used as a quantitative assay system for chemical and physical carcinogens. 10T cells, but not their transformed counterparts, exhibit a decreased final saturation density with decreasing serum concentration. Exposure of carcinogen-treated cultures to 5% serum 8 days posttreatment led to a 2- to 6-fold enhancement in transformation frequency in comparison with cultures maintained in 10% serum throughout. Exposure 14, 21, or 28 days posttreatment also caused enhancement of transformation frequency, provided a sufficient time for expression of the malignant phenotype was allowed. Exposure to 5% serum 1 or 2 days posttreatment did not lead to significant enhancement of transformation frequency. In contrast, exposure to 15 or 20% serum after 8 days virtually abolished the expression of malignancy; however, this inhibition could be reversed by 5% serum. Morphologically transformed foci isolated from cultures exposed to 5% serum produced clones in agarose with the same frequency as did foci isolated from cultures exposed to 10% serum.

Reconstruction experiments, utilizing confluent monolayers of 10T cells overlaid with transformed cells, demonstrated that the growth of transformed cells decreased proportionally with the log of serum concentration. This effect was not caused by depletion of medium and was dependent upon the presence of 10T cells. It is concluded that the expression of malignancy in this system is governed by the serum-modulated cell density of the mass of nontransformed cells in the culture.

¹ Supported in part by Grant CA 13038 from the National Cancer Institute, USPHS.

Received 6/16/76. Accepted 11/12/76.

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