Isolation of a differentiation‐defective myoblastic cell line, INC2, expressing muscle LIM protein under differentiation‐inducing conditions

A non-differentiating myoblastic cell line, INC2, and a differentiating cell line, COM3, were established from the mouse myoblastic cell line C2C12. Under differentiation conditions, both COM3 and INC2 cells stopped proliferation in a similar manner. The COM3 cells then differentiated into myotubes during the 4-day differentiation culture. In contrast, almost none of the INC2 cells differentiated into myotubes even in differentiation medium. Reverse transcriptase-polymerase chain reaction (RT-PCR) and immunoblot analyses showed that the levels of myogenin and MyoD proteins were significantly decreased in INC2 cells. The differentiation marker sarcomeric myosin heavy chain (MHC) was expressed in COM3 but not in INC2 cells. In contrast, both INC2 and COM3 cells expressed another myogenic regulatory factor, muscle LIM protein (MLP), in a differentiation condition-dependent manner. These results suggest that MLP gene expression is regulated in a myogenin/MyoD-independent manner. Enforced expression of the myogenin gene induced MHC expression in INC2 cells. Thus, the signaling pathway situated downstream is assumed to be intact in INC2 cells and suppression of myogenin, gene expression may be a primary defect in INC2 cells.