Kurabayashi M, Jeyaseelan R, Kedes L
Department of Biochemistry and Molecular Biology, University of Southern California School of Medicine, Los Angeles 90033.
J Biol Chem. 1994 Feb 25;269(8):6031-9.
We have shown recently that Doxorubicin (Dox, Adriamycin), a potent broad spectrum chemotherapeutic agent with a major side effect of cardiomyopathy, completely prevents myoblast fusion and accumulation of muscle-specific transcripts in the mouse C2 skeletal muscle cell line. Here we use mouse embryonic fibroblast 10T1/2 cells to demonstrate that Dox represses muscle-specific gene expression by interfering with MyoD activity. As assayed by transient cotransfection, Dox inhibits the ability of MyoD to trans-activate muscle-specific reporter genes. A stable cell system was developed in which MyoD is constitutively expressed in 10T1/2 cells (M10 cells). Dox-treated M10 cells express MyoD from a long terminal repeat-driven vector but fail to activate endogenous MyoD and myogenin loci. Dox did not effect E2A gene transcript levels, but Id mRNA levels are significantly increased in Dox-treated M10 cells. Interestingly, overexpression of E2-5, which forms inactive heterodimers with Id, can overcome the Dox-induced suppression of the trans-activation function of MyoD in 10T1/2 cells. Furthermore, we demonstrate that the 5'-flanking region of the Id2 gene mediates its Dox-inducible transcriptional expression. These findings support a model in which Dox inhibits muscle-specific gene expression by interfering with the function of MyoD protein through, at least in part, induction of Id gene expression. The implications of our results for the molecular mechanisms underlying the myofibrillar loss observed in Dox-induced cardiomyopathy are discussed.
我们最近发现，阿霉素（Dox，多柔比星）是一种具有心肌病这一主要副作用的强效广谱化疗药物，它能完全阻止小鼠C2骨骼肌细胞系中的成肌细胞融合以及肌肉特异性转录本的积累。在此，我们利用小鼠胚胎成纤维细胞10T1/2来证明阿霉素通过干扰MyoD的活性来抑制肌肉特异性基因表达。通过瞬时共转染检测，阿霉素抑制了MyoD反式激活肌肉特异性报告基因的能力。我们建立了一个稳定的细胞系统，其中MyoD在10T1/2细胞（M10细胞）中组成性表达。经阿霉素处理的M10细胞从长末端重复序列驱动的载体表达MyoD，但未能激活内源性MyoD和肌细胞生成素基因座。阿霉素不影响E2A基因转录水平，但在经阿霉素处理的M10细胞中，Id mRNA水平显著升高。有趣的是，与Id形成无活性异二聚体的E2-5的过表达能够克服阿霉素诱导的对10T1/2细胞中MyoD反式激活功能的抑制。此外，我们证明Id2基因的5'侧翼区域介导其阿霉素诱导的转录表达。这些发现支持了一个模型，即阿霉素通过至少部分地诱导Id基因表达来干扰MyoD蛋白的功能，从而抑制肌肉特异性基因表达。我们讨论了我们的结果对于阿霉素诱导的心肌病中观察到的肌原纤维损失的分子机制的意义。
