Further Characterization of Bc3h1 Myogenic Cells Reveals Lack of P53 Activity and Underexpression of Several P53 Regulated and Extracellular Matrix–associated Gene Products

Abstract

To catalog factors that may contribute to the completion of myogenesis, we have been looking for molecular differences between BC3H1 and C2C12 cells. Cells of the BC3H1 tumor line, though myogenic, are nonfusing, and withdraw from the cell cycle only reversibly, whereas cells of the C2C12 line fuse, differentiate terminally, and express several muscle-specific gene products that BC3H1 cells do not. Relative to C2C12 cells, BC3H1 cells underaccumulated cyclin-dependent kinase inhibitor p21 and underaccumulated transcripts for p21, GADD45, CDO, decorin, osteopontin, H19, fibronectin, and thrombospondin-1 (tsp-1). Levels of accumulation of H19, tsp-1, and larger isoforms of fibronectin messenger ribonucleic acid (mRNA) were found to increase in response to expression of myogenic regulatory factors as shown by their accumulation in differentiated myogenically converted 10T1/2 cells but not in 10T1/2 fibroblasts. BC3H1s accumulated a temperature-insensitive, geldanamycin-sensitive, misfolded form of p53 incapable of transactivating a p53 responsive reporter, consistent with underexpression of p21, GADD45, and tsp-1. BC3H1 and C2C12 cells were similar with respect to upregulation of p27 protein, downregulation of mitogen-activated protein kinase phosphatase-1 (MKP-1) protein, upregulation of retinoblastoma (Rb) mRNA, and nuclear localization of hypophosphorylated Rb. Cells of both lines expressed the muscle-specific 1b isoform of MEF2D. Although nonfusing in the short term, after more than 18 d in differentiation medium, some cultures of BC3H1 cells formed viable multinucleated cells in which the nuclei did not reinitiate synthesis of DNA in response to serum. Our findings suggest participation of tsp-1 and specific isoforms of fibronectin in myogenesis and suggest additional avenues of research in myogenesis and oncogenesis.

Identification of genes differentially expressed during prenatal development of skeletal muscle in two pig breeds differing in muscularity

“…SPP1 is a target of MYOD and MYF5 [ 33 ] and was shown to be expressed in vitro in myoblasts and myotubes, with higher levels in the later [ 34 ]. Moreover SPP1 is downregulated in the BC3H1 cell line, which is myogenic but do not terminally differentiate, compared to the myogenic C2C12 cell line, which differentiate normally [ 35 ]. Considering our in vivo and the in vitro evidences for an association between SPP1 expression and myogenic differentiation it appears that upregulation of SPP1 in Duroc may be a sign of accelerated myogenesis in this breed compared to Pietrain.…”

Terminal Differentiation of Myogenic Cells Requires Stable Form of Tumor Suppressor Protein, P53

“…Four myogenic regulatory factors (MyoD, myogenin, Myf5, and MRF4) and MEF2 play a central role during conversion of myoblasts into myotubes (14,17,19,30) and constitutive expression of any of these factors in fibroblasts turns on the myogenic cascade pathway (14,17,19,30). However, a nonfusing cell line BC3H1 (smooth muscle like brain tumor), which exhibits properties of smooth and skeletal muscles, contains functionally inactive MyoD but undergoes differentiation (28). To examine whether the expression of p53 is MyoD dependent while BC3H1 cells were undergoing differentiation, these cells were cultured and allowed to differentiate in differentiation medium.…”

“…On the other hand, BC3H1 myogenic cells, which reversibly withdraw from cell cycle and differentiate into non-fusing cells. In these cells, there is no functional MyoD, although, they express Myf5, myogenin, MRF4, MEF2, and several other muscle-specific genes (28). p53's roles in differentiation are very puzzling.…”

“…Differentiation in culture of mouse C2C12 myoblasts, which are originated from leg thigh muscles, is an excellent model to study molecular details of myogenesis during early differentiation and late terminal differentiation (26). While BC3H1 myogenic cells derived from brain tumor undergo myogenesis but do not permanently withdraw from cell cycle and show terminal differentiation (27)(28)(29). We therefore examined the cytoplasmic and nuclear levels of p53 in these two cell lines undergoing differentiation.…”