SKELETAL-MUSCLE CELLS LACKING THE RETINOBLASTOMA PROTEIN DISPLAY DEFECTS IN MUSCLE GENE-EXPRESSION AND ACCUMULATE IN S-PHASES AND G(2)-PHASES OF THE CELL-CYCLE

Viral oncoproteins that inactivate the retinoblastoma tumor suppressor protein (pRb) family both block skeletal muscle differentiation and p romote cell cycle progression. To clarify the dependence of terminal d ifferentiation on the presence of the different pRb-related proteins, we have studied myogenesis using isogenic primary fibroblasts derived from mouse embryos individually deficient for pRb, p107, or p130. When ectopically expressed in fibroblasts lacking pRb, MyoD induces an abe rrant skeletal muscle differentiation program characterized by normal expression of early differentiation markers such as myogenin and p21, but attenuated expression of late differentiation markers such as myos in heavy chain (MHC). Similar defects in MHC expression were not obser ved in cells lacking either p107 or p130, indicating that the defect i s specific to the loss of pRb. In contrast to wild-type, p107-deficien t, or p130-deficient differentiated myocytes that are permanently with drawn from the cell cycle, differentiated myocytes lacking pRb accumul ate in S and G(2) phases and express extremely high levels of cyclins A and B, cyclin-dependent kinase (Cdk2), and Cdc2, but fail to readily proceed to mitosis. Administration of caffeine, an agent that removes inhibitory phosphorylations on inactive Cdc2/cyclin B complexes, spec ifically induced mitotic catastrophe in pRb-deficient myocytes, consis tent with the observation that the majority of pRb-deficient myocytes arrest in S and G(2) Together, these findings indicate that pRb is req uired for the expression of late skeletal muscle differentiation marke rs and for the inhibition of DNA synthesis, but that a pRb-independent mechanism restricts entry of differentiated myocytes into mitosis.