CYCLINS AND CYCLIN-DEPENDENT KINASES ARE DIFFERENTIALLY REGULATED DURING TERMINAL DIFFERENTIATION OF C2C12 MUSCLE-CELLS

Differentiation of skeletal myoblasts into contractile myotubes is ass ociated with permanent withdrawal from the cell cycle. Little is known about the expression of cell cycle regulating genes during terminal d ifferentiation of muscle cells. We investigated the expression pattern , biological activity, and cellular localization of cyclins and cyclin -dependent kinases during terminal differentiation of the mouse skelet al myogenic cell line C2C12. After induction of differentiation by ser um deprivation, cdc2 mRNA levels transiently increased, followed by a down-regulation to undetectable levels within 42 h. In contrast, cdk2 mRNA stayed constant during this period. During differentiation cyclin A, B, and C were down-regulated within 24 h to undetectable levels. I nterestingly, cyclin D1/CYL1 mRNA was upregulated by twofold at 9-12 h after serum deprivation followed by a down-regulation to undetectable levels within 42 h, while cyclin D3/CYL3 mRNA levels remained constan t. Restimulation of the differentiated myotube culture with serum rein duced cdc2 as well as cyclin D1/CYL1 mRNA close to the levels observed in dividing myoblasts. At the protein level p34(cdc2) was detected in nuclei of proliferating myoblasts and nascent myotubes, but not in ma ture myotubes. Restimulation with serum-induced p34(cdc2) protein in a small minority of unfused myoblasts, but never in myotubes. Histone H 1 kinase activity of p34(cdc2) decreased during differentiation while p33(cdk2) activity did not change. These findings suggest that termina l differentiation of skeletal muscle cells is associated with a differ ential regulation of cyclins and their associated kinases. Inability t o accumulate p34(cdc2) protein in response to serum stimulation, despi te the induction of its mRNA, in differentiated myotubes may play an i mportant role in maintaining the postmitotic state of skeletal muscle in the presence of high concentrations of growth factors. (C) 1994 Aca demic Press, Inc.