Role of cytoskeleton changes and expression of the H-ras oncogene during promotion of neoplastic transformation in mouse epidermal JB6 cells.

The relationship between cytoskeletal changes and oncogene expression in initiated cells during exposure to a tumor promoter was investigated in the phorbol ester-sensitive murine epidermis-derived cell line JB6 (P+ cells) and its promotion-insensitive variant (P- cells) using immunocytochemical methods, soft agar assays, and tumorigenicity tests in nude mice. Cytoskeletal changes in P+ and P- cells induced by short-term incubation with 12-O-tetradecanoylphorbol-13-acetate (TPA) were similar. Prolonged incubation with TPA allowed P- cells to regain their original appearance and resulted in growth inhibition; however, the extended presence of TPA produced in P+ cells persistent alterations in the distribution of actin, vinculin, and fibronectin. P+ cells proceeded to develop multilayered foci. Using monoclonal antibodies, we detected the H-ras oncogene-encoded Mr 21,000 protein (p21) exclusively in focus-forming cells. Both the observed morphological changes and the expression of p21 were reversible in P+ cells when TPA exposure was terminated soon after foci had developed. In order for TPA-treated P+ cells to grow as tumors in nude mice, multiple cycles of exposure to TPA in conjunction with clonal expansion in agar were necessary. The results indicate that there exists during promotion of the P+ JB6 cells a relationship between expression of the H-ras gene product p21 and enhanced proliferation with focus formation and that both expression of p21 and focus formation depend on the continuous presence of the promoting agent.