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Enhanced Induction of the Anchorage-independent Phenotype in Initiated Rat Tracheal Epithelial Cell Cultures by the Tumor Promoter 12-O-Tetradecanoylphorbol-13-acetate

Laboratory of Pulmonary Function and Toxicology, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709

The purpose of the studies reported here was to compare the response of noninitiated and initiated primary rat tracheal epithelial (RTE) cell cultures to the mouse skin tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). The endpoints measured were number of cells per culture, colony-forming efficiency, subculturability, and colony formation in soft agarose. Primary RTE cell cultures were exposed on Day 1 to either 0.2% dimethyl sulfoxide, or to 0.1 µg per ml of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Thereafter, the same cultures were exposed twice weekly from Days 6 to 30 to either 0.2% dimethyl sulfoxide or to TPA (10 pg/ml). Sequential exposure to MNNG and TPA did not increase the number of viable cells per culture beyond that seen in MNNG-exposed cultures. Determination of the frequency of colony-forming cells 10 days after the end of the initiation-promotion treatment (Day 40 of culture) revealed a marked enhancement in colony-forming efficiency of treated cultures compared to dimethyl sulfoxide-exposed control cultures. However, sequential exposure to MNNG and TPA had an additive or slightly more than additive effect on the colony-forming efficiency of RTE cells exposed to MNNG or TPA only.

Treatment of the primary cultures with MNNG alone or TPA alone increased the subculturability of RTE cells to a similar extent. The sequential exposure to MNNG followed by TPA appeared to have an additive effect on the frequency of subcul-turability.

The most pronounced effect of the sequential MNNG-TPA exposure as compared to single-agent exposure was a marked enhancement of the anchorage-independent (ag⁺) phenotype. Of the cultures treated with MNNG followed by TPA, over 50% were ag⁺ at 60 days. In contrast, of the cultures treated either with MNNG alone or with TPA alone, only 3% were ag⁺ on Day 60. (All control cultures were ag^-.) Colony-forming efficiency in soft agarose also increased disproportionately between 60 and 120 days in initiated-promoted cultures.

These experiments indicate that the major effect of the tumor promoter TPA on initiated RTE cell cultures is to enhance the appearance of the late ag⁺-phenotype.

¹ To whom requests for reprints should be addressed, at Northrop Services, Inc., Box 12313, Research Triangle Park, NC 27709.

Received 3/13/84. Accepted 7/27/84.