Rapid Activation and Down-Regulation of Protein Kinase C a in 12-O-Tetradecanoyl phorbol-13-acetate-induced Differentiation of Human Rhabdomyosarcoma Cells.

Human rhabdomyosarcoma RD cells express the myogenic regulatory fadors MyoD and myogenin but differentiate spontaneously very poorly. Prolonged treatment of RD cells with the protein kinase C (PKC) adivator 1 2-O-tetradecanoylphorbol-1 3-acetate (TPA) induces growth arrest and myogenic differentiation as shown by the accumulation of a-adin and myosin light and heavy chains, without affeding the expression of MyoD and myogenin. In this study, we show that shortterm phorbol ester treatment of the cultures is sufficient to trigger myogenic differentiation but not growth arrest. Furthermore, PKC inhibitors, such as staurosporine or calphostin C, prevent TPA-induced differentiation but not cell growth arrest. These data suggest that the two events are mediated by different pathways; a possible interpretation is that the adivation of one or more PKC isoforms mediates the indudion of differentiation, whereas the down-regulation of the same or different isoforms mediates the growth arrest. To address the mechanism whereby TPA affeds cell growth and differentiation in RD cells, we first analyzed PKC isoenzyme distribution. We found that RD cells express the a, ıi1, 7, and ı PKC isoenzymes. Only the a isoform is exclusively found in the soluble fradion, but it translocates to the membrane fradion within 5 mm of TPA treatment and is completely down-regulated after 6 h. The other isoenzymes are found associated to both the soluble and the particulate fradions and are downregulated after long-term TPA treatment. By immunofluorescence analysis, we show that the PKC a down-regulation is specific for those cells that respond to TPA by adivating the muscle phenotype. We propose that TPA-induced differentiation in RD cells is mediated by the transient adivation of PKC a, which adivates some of the intracellular events that are necessary for MyoD and myogenin transading adivity and for the indudion of terminal differentiation of RD cells. By contrast, the constitutively adive ı and ı are responsible for the maintenance of cell growth, and their down-regulation is responsible for long-term TPA-induced cell growth arrest.