RAPID ACTIVATION AND DOWN-REGULATION OF PROTEIN-KINASE-C-ALPHA IN 12-O-TETRADECANOYLPHORBOL-13-ACETATE-INDUCED DIFFERENTIATION OF HUMAN RHABDOMYOSARCOMA CELLS

Human rhabdomyosarcoma RD cells express the myogenic regulatory factor s MyoD and myogenin but differentiate spontaneously very poorly. Prolo nged treatment of RD cells with the protein kinase C (PKC) activator 1 2-O-tetradecanoylphorbol-13-acetate (TPA) induces growth arrest and my ogenic differentiation as shown by the accumulation of alpha-actin and myosin light and heavy chains, without affecting the expression of My oD and myogenin. In this study, we show that shortterm phorbol ester t reatment of the cultures is sufficient to trigger myogenic differentia tion but not growth arrest. Furthermore, PKC inhibitors, such as staur osporine or calphostin C, prevent TPA-induced differentiation but not cell growth arrest. These data suggest that the two events are mediate d by different pathways; a possible interpretation is that the activat ion of one or more PKC isoforms mediates the induction of differentiat ion, whereas the down-regulation of the same or different isoforms med iates the growth arrest. To address the mechanism whereby TPA affects cell growth and differentiation in RD cells, we first analyzed PKC iso enzyme distribution. We found that RD cells express the alpha, beta(1) , gamma, and zeta PKC isoenzymes. Only the alpha isoform is exclusivel y found in the soluble fraction, but it translocates to the membrane f raction within 5 min of TPA treatment and is completely down-regulated after 6 h. The other isoenzymes are found associated to bath the solu ble and the particulate fractions and are downregulated after lone-ter m TPA treatment. By immunofluorescence analysis, we show that the PKC alpha down-regulation is specific for those cells that respond to TPA by activating the muscle phenotype. We propose that TPA-induced differ entiation in RD cells is mediated by the transient activation of PKC a lpha, which activates some of the intracellular events that are necess ary for MyoD and myogenin transacting activity and for the induction o f terminal differentiation of RD cells. By contrast, the constitutivel y active beta(1) and zeta are responsible for the maintenance of cell growth, and their down-regulation is responsible for long-term TPA-ind uced cell growth arrest.