Commitment, fusion and biochemical differentiation of a myogenic cell line in the absence of DNA synthesis

Abstract

L6E9 rat myoblasts derived from the L6 cell line can be induced to differentiate to a very high percentage by manipulating the culture conditions. Under standard differentiating conditions, L6E9 cells divide an average of 2.5 times before differentiating and > 99% of them incorporate 3H-TdR [thymidine] before fusing. By inhibiting DNA replication it was demonstrated that DNA synthesis is not required to switch from growth to differentiation. After every cell division, L6E9 cells can fuse or proliferate without intervening DNA synthesis. Cell cloning and DNA labeling experiments show a direct correlation between the time of culture in differentiating medium and a progressive loss of proliferative capacity of mononucleated L6E9 cells, demonstrating that these cells become irreversibly committed to differentiation and withdraw from the cell cycle prior to and not as a consequence of cell fusion. The commitment step occurs during the G1 phase prior to fusion. This G1 phase has a latent period during which no irreversible step toward differentiation occurs and the cells remain ambivalent toward growth or differentiation. Under proper conditions, this period is followed by an irreversible commitment toward differentiation and a loss of proliferative capacity. The kinetics of this commitment step strongly suggest that L6E9 cells become irreversibly committed in a stochastic manner. Once the cells have become committed, with or without DNA synthesis, they will fuse to form myotubes and biochemically differentiate in a deterministic fashion. A stochastic model of differentiation for L6E9 cells is supported. The switch from a proliferating to a differentiating genetic program can apparently occur in the absence of DNA synthesis.