Miller Patrick J, Hollenbach Andrew D
Department of Genetics, Louisiana State University Health Sciences Center, 533 Bolivar Street, New Orleans, LA 70112, USA.
Biochim Biophys Acta. 2007 Oct;1770(10):1450-8. doi: 10.1016/j.bbagen.2007.06.016. Epub 2007 Jul 13.
The childhood solid muscle tumor Alveolar Rhabdomyosarcoma (ARMS) is characterized by the t(2;13)(q35;q14) chromosomal translocation, which results in the fusion of two transcription factors important for myogenesis, Pax3 and FKHR (FOX01a). The effects of myogenic differentiation on the stability of FKHR have been well characterized. However, similar studies have yet to be performed on Pax3 or the oncogenic fusion protein Pax3-FKHR. Therefore, we demonstrate in the physiologically relevant mouse primary myoblast system that the expression of Pax3 decreases nearly 95% during the first 24 h of myogenic differentiation. In contrast, there is an aberrant persistence of expression of Pax3-FKHR during this same time period. These differences in protein expression levels do not result from changes on the transcriptional nor the translational level since we observed no concomitant decrease in the levels of Pax3 or Pax3-FKHR mRNA or in the ability of both proteins to be translated. Instead, a pulse-chase analysis determined that Pax3-FKHR has a half-life significantly greater than\ the half-life of wild type Pax3 demonstrating for the first time that Pax3-FKHR has greater post-translational protein stability relative to wild type Pax3 during early myogenic differentiation. Finally, the persistence of expression of Pax3-FKHR prevents the terminal differentiation of primary myoblasts demonstrating a biological consequence of its aberrant expression.
儿童实体肌肉肿瘤肺泡横纹肌肉瘤（ARMS）的特征是存在t(2;13)(q35;q14)染色体易位，这导致了两个对肌生成很重要的转录因子Pax3和FKHR（FOX01a）的融合。肌源性分化对FKHR稳定性的影响已得到充分表征。然而，尚未对Pax3或致癌融合蛋白Pax3-FKHR进行类似研究。因此，我们在生理相关的小鼠原代成肌细胞系统中证明，在肌源性分化的最初24小时内，Pax3的表达下降了近95%。相比之下，在同一时期，Pax3-FKHR的表达却异常持续。这些蛋白质表达水平的差异并非源于转录或翻译水平的变化，因为我们观察到Pax3或Pax3-FKHR mRNA水平没有相应下降，且这两种蛋白质的翻译能力也未受影响。相反，脉冲追踪分析确定Pax3-FKHR的半衰期明显长于野生型Pax3的半衰期，这首次表明在早期肌源性分化过程中，相对于野生型Pax3，Pax3-FKHR具有更高的翻译后蛋白质稳定性。最后，Pax3-FKHR表达的持续阻止了原代成肌细胞的终末分化，证明了其异常表达的生物学后果。
