Compton R S, Konigsberg I R
Department of Biology, University of Virginia, Charlottesville 22901.
Dev Biol. 1988 Oct;129(2):476-94. doi: 10.1016/0012-1606(88)90394-6.
Skeletal muscle differentiation is accompanied by the withdrawal of the proliferating myoblasts from the cell cycle in the G1 phase. We showed earlier that the length of G1 and the timing of the differentiative transition could be controlled in large part by the composition of the culture medium. In this study we have asked whether a G1 arrest imposed independently of the culture medium is sufficient to elicit the differentiative response. To examine this possibility we have characterized a new G1-specific ts murine myoblast line. This line, ts-36, was identified as a G1-specific mutant on the basis of four criteria: prolonged viability at the nonpermissive temperature (npt), the kinetics of cell cycle withdrawal and reentry in temperature shift experiments, the ability of the cells to differentiate at the npt in low-growth medium, and, finally, the observation that, by the criterion of flow microfluorometry, the mutant cells block at the G1 landmark in the cell cycle. A ts-imposed G1 arrest of up to 96-hr duration is by itself insufficient to activate the differentiative program in ts-36 cells cultured in complete growth medium. The differentiated phenotype is expressed, however, in temperature-arrested cells cultured either in low-growth (conditioned) medium or in a medium from which mitogens have been removed by ultrafiltration. Differentiation can be reversed by refeeding with complete growth medium. The effects of growth medium can be mimicked by FGF to the extent of inhibiting activation of the differentiative program in temperature-arrested ts-36 cells and in eliciting downregulation of muscle-specific contractile protein synthesis. Extrapolating from these observations suggests that growth factors may have more than one role in myogenesis in vitro. They not only stimulate proliferation, but also inhibit differentiation in the absence of proliferation. Examining the kinetics of withdrawal from the cell cycle indicates that ts-36, cultured in conditioned medium blocks at the npt restriction point rather than the conditioned medium block. Our results suggest that two conditions must be met to trigger myogenic differentiation in vitro. Withdrawal from the cell cycle in G1 alone is not sufficient. Reduction of the mitogen level in the medium below a threshold level is an obligate condition for phenotypic expression.
骨骼肌分化伴随着增殖性成肌细胞在细胞周期的G1期退出细胞周期。我们之前表明，G1期的长度和分化转变的时间在很大程度上可以由培养基的组成来控制。在本研究中，我们探讨了独立于培养基施加的G1期阻滞是否足以引发分化反应。为了检验这种可能性，我们鉴定了一种新的G1期特异性温度敏感型小鼠成肌细胞系。这个细胞系，ts - 36，基于四个标准被鉴定为G1期特异性突变体：在非允许温度（npt）下延长的存活能力、温度转换实验中细胞周期退出和重新进入的动力学、细胞在低生长培养基中在npt时分化的能力，以及最后，通过流式细胞荧光测定法的标准观察到，突变细胞在细胞周期的G1标记处阻滞。在完全生长培养基中培养的ts - 36细胞中，长达96小时的温度敏感型G1期阻滞本身不足以激活分化程序。然而，在低生长（条件性）培养基中或通过超滤去除有丝分裂原的培养基中培养的温度阻滞细胞中表达了分化表型。通过重新添加完全生长培养基可以使分化逆转。生长培养基的作用可以被FGF模拟，在抑制温度阻滞的ts - 36细胞中分化程序的激活以及引发肌肉特异性收缩蛋白合成的下调方面。从这些观察结果推断表明，生长因子在体外肌生成中可能具有不止一种作用。它们不仅刺激增殖，而且在没有增殖的情况下抑制分化。检查从细胞周期退出的动力学表明，在条件性培养基中培养的ts - 36在npt限制点阻滞，而不是在条件性培养基阻滞。我们的结果表明，在体外触发成肌分化必须满足两个条件。仅在G1期退出细胞周期是不够的。将培养基中有丝分裂原水平降低到阈值水平以下是表型表达的必要条件。
