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Four-Day Duration of Tumor Promoter Exposure Required to Transform JB6 Promotion-sensitive Cells to Anchorage Independence

Laboratory of Viral Carcinogenesis [L. D. D., N. H. C.] and Laboratory of Experimental Pathology [T. D. G.], National Cancer Institute, Frederick, Maryland 21701

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 M_r 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.

¹ Present address: Department of Physiology and Biophysics, University of Alabama at Birmingham, Birmingham, AL 35233.

² To whom requests for reprints should be addressed, at Laboratory of Viral Carcinogenesis, National Cancer Institute, Frederick Cancer Research Facility, Building 560, Room 21-89, Frederick, MD 21701.

Received 2/ 5/88. Revised 6/25/88. Revised 9/15/88. Accepted 9/19/88.