Four-day duration of tumor promoter exposure required to transform JB6 promotion-sensitive cells to anchorage independence.

The JB6 mouse epidermal cell lines have been developed to study promotion of neoplastic transformation in vitro. Treatment of JB6 cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) at 1 to 100 ng/ml results in the irreversible acquisition of tumorigenicity in nude mice and anchorage-independent growth. Among the biochemical responses which occur during TPA treatment is a decrease in procollagen synthesis. During a study of the possible role of H2O2 in the process of promotion, it was observed that catalase purchased commercially would inhibit TPA promotion as well as the reduction of collagen synthesis in a dose-dependent manner. A highly purified catalase preparation failed to demonstrate the TPA blocking activity, suggesting that this activity was due to a contaminating factor. We have separated the TPA blocking factor from the catalase itself using concanavalin A affinity chromatography. The factor is a Mr 60,000 glycoprotein showing TPA hydrolase activity. The enzyme, which is similar to a murine liver-derived TPA hydrolase, produces a single phorbol product from TPA that has been identified as phorbol-13-acetate. TPA hydrolase was used to terminate TPA action in soft agar. This made it possible to establish that approximately 4 days of exposure to phorbol esters are required for promotion of transformation in the JB6 model system.