EXPRESSION OF THE POSITIVE REGULATOR OF CELL-CYCLE PROGRESSION, CYCLIN D3, IS INDUCED DURING DIFFERENTIATION OF MYOBLASTS INTO QUIESCENT MYOTUBES

L(6) cells are committed skeletal muscle precursors which can be induc ed to differentiate into multinucleated, terminally differentiated myo tubes. Upon differentiation, these immature skeletal myotubes enter a quiescent state and are unable to reenter the cell cycle. We have exam ined expression of a series of genes involved in regulation of progres sion through the G(1)/S boundary in undifferentiated L(6) cells and du ring terminal differentiation of L(6) myoblasts. While no change in th e level of cyclin D1 transcript and a transient increase in cyclin D2 transcript were observed, a large increase in cyclin D3 expression was found. Immunohistochemistry demonstrated strong staining for cyclin D 3 protein in the nuclei of the multinucleated myotubes from 4 independ ent myoblast cell lines; L(6), L(8), G(8) and C2C12. Immunoprecipitati on confirmed a greater than 20-fold increase in the levels of cyclin D 3 protein in the differentiated L(6) myotubes as well as its associati on with a number of proteins. Western assays demonstrated, further, th at cyclin D3 was complexed with the cyclin dependent-kinases, cdk2 and cdk4, in differentiated L(6) cells. However, while kinase activity sp ecific for a GST-pRB fusion protein was seen for cyclin D3-containing complexes isolated from undifferentiated cells, the high levels of cyc lin D3 in the differentiated myotubes had no associated kinase activit y. These data demonstrate that cyclin D3 may also have a function in t erminally differentiated, quiescent cells. The lack of cyclin D3-assoc iated kinase activity and its association with a number of different p roteins suggest that cyclin D3 may regulate the function of other prot eins by direct interaction with these factors.