Shields Janiel M, Mehta Heena, Pruitt Kevin, Der Channing J
Department of Pharmacology, Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, 27599, USA.
Mol Cell Biol. 2002 Apr;22(7):2304-17. doi: 10.1128/MCB.22.7.2304-2317.2002.
We showed previously that activated Ras, but not Raf, causes transformation of RIE-1 epithelial cells, demonstrating the importance of Raf-independent pathways in mediating Ras transformation. To assess the mechanism by which Raf-independent effector signaling pathways contribute to Ras-mediated transformation, we recently utilized representational difference analysis to identify genes expressed in a deregulated fashion by activated Ras but not Raf. One gene identified in these analyses encodes for alpha-tropomyosin. Therefore, we evaluated the mechanism by which Ras causes the downregulation of tropomyosin expression. By using RIE-1 cells that harbor inducible expression of activated H-Ras(12V), we determined that the downregulation of tropomyosin expression correlated with the onset of morphological transformation. We found that the reversal of Ras transformation caused by inhibition of extracellular signal-regulated kinase activation corresponded to a restoration of tropomyosin expression. Inhibition of p38 activity in Raf-expressing RIE-1 cells caused both morphological transformation and loss of tropomyosin expression. Thus, a reduction in tropomyosin expression correlated strictly with morphological transformation of RIE-1 cells. However, forced overexpression of tropomyosin in Ras-transformed cells did not reverse morphological or growth transformation, a finding consistent with the possibility that multiple changes in gene expression contribute to Ras transformation. We also determined that tropomyosin expression was low in two human tumor cell lines, DLD-1 and HT1080, that harbor endogenous mutated alleles of ras, but high in transformation-impaired, derivative cell lines in which the mutant ras allele has been genetically deleted. Finally, treatment with azadeoxycytidine restored tropomyosin expression in Ras-transformed RIE-1, HT1080, and DLD-1 cells, suggesting a role for DNA methylation in downregulating tropomyosin expression.
我们之前表明，激活的Ras而非Raf可导致RIE-1上皮细胞发生转化，这证明了Raf非依赖途径在介导Ras转化中的重要性。为了评估Raf非依赖效应信号通路促进Ras介导的转化的机制，我们最近利用代表性差异分析来鉴定由激活的Ras而非Raf以失调方式表达的基因。在这些分析中鉴定出的一个基因编码α-原肌球蛋白。因此，我们评估了Ras导致原肌球蛋白表达下调的机制。通过使用携带可诱导表达激活型H-Ras(12V)的RIE-1细胞，我们确定原肌球蛋白表达的下调与形态转化的开始相关。我们发现，抑制细胞外信号调节激酶激活所导致的Ras转化的逆转与原肌球蛋白表达的恢复相对应。在表达Raf的RIE-1细胞中抑制p38活性会导致形态转化和原肌球蛋白表达的丧失。因此，原肌球蛋白表达的降低与RIE-1细胞的形态转化严格相关。然而，在Ras转化细胞中强制过表达原肌球蛋白并不能逆转形态或生长转化，这一发现与基因表达的多种变化促成Ras转化的可能性一致。我们还确定，在携带内源性ras突变等位基因的两个人类肿瘤细胞系DLD-1和HT1080中，原肌球蛋白表达较低，但在突变ras等位基因已被基因删除的转化受损的衍生细胞系中表达较高。最后，用氮杂脱氧胞苷处理可恢复Ras转化的RIE-1、HT1080和DLD-1细胞中的原肌球蛋白表达，这表明DNA甲基化在下调原肌球蛋白表达中起作用。
