Bazarov A V, Adachi S, Li S F, Mateyak M K, Wei S, Sedivy J M
Department of Molecular Biology, Cell Biology, and Biochemistry, Brown University, Providence, Rhode Island 02912, USA.
Cancer Res. 2001 Feb 1;61(3):1178-86.
Dergulation of c-myc and mutation of ras genes is commonly found in many human tumors. Several lines of evidence indicate that c-Myc and oncogenic Ras cooperate in causing malignant transformation, but the mechanism of this cooperation is not understood. We set out to investigate the effect on transformation of a modest reduction in endogenous c-Myc expression, which was achieved using a c-myc heterozygous cell line constructed by targeted homologous recombination. In contrast to previous reports where c-Myc expression or activity was ablated using antisense or dominant-defective methods, use of c-myc +/- cells provides a stable and homogeneous cell culture system with a precisely defined c-Myc expression level. In addition, this approach does not suffer from nonspecific artifacts such as antisense oligonucleotide toxicity or interference of dominant-defective proteins with multiple (and often undefined) target proteins. The striking and unexpected finding communicated here is that the relatively modest 50% reduction in c-Myc expression resulted in a greater than 10-fold reduction in susceptibility to transformation by oncogenic Ras or Raf proteins. This very significant defect in transformation potential cannot be explained on the basis of a generalized cell-cycle defect, because c-myc +/- cells exhibit only a minimal (20%) reduction in proliferation. Genetic epistasis analysis indicated that c-Myc and Ras acted by independent pathways that converged to regulate the abundance of the cyclin-dependent kinase inhibitor protein p27Kip1. Anchorage deprivation elicited a strong up-regulation of p27, and a 50% reduction in c-Myc expression significantly compromised the ability of Ras to down-regulate p27. We propose that Ras and c-Myc signals cooperate to regulate the activity of cyclin D-Cdk4/6 complexes: the former by up-regulating the expression of cyclin D1 and the latter by affecting the activity of the complexes. Ectopic expression of cyclin A restored the transformation potential of c-myc +/- cells, implicating it as a downstream genetic component in the pathway. From a therapeutic standpoint, it is of interest that, although transformation appears to be very sensitive to c-Myc expression levels, much larger reductions can be tolerated without causing any significant cell cycle defects.
c-myc基因的失调和ras基因的突变在许多人类肿瘤中普遍存在。多项证据表明，c-Myc和致癌性Ras在导致恶性转化过程中相互协作，但其协作机制尚不清楚。我们着手研究内源性c-Myc表达适度降低对转化的影响，这是通过使用靶向同源重组构建的c-myc杂合细胞系实现的。与之前使用反义或显性缺陷方法消除c-Myc表达或活性的报道不同，使用c-myc+/-细胞提供了一个稳定且均一的细胞培养系统，其c-Myc表达水平精确界定。此外，这种方法不会受到非特异性假象的影响，如反义寡核苷酸毒性或显性缺陷蛋白对多种（通常未明确）靶蛋白的干扰。此处传达的惊人且意外的发现是，c-Myc表达相对适度降低50%会导致致癌性Ras或Raf蛋白诱导转化的易感性降低超过10倍。这种转化潜能的显著缺陷不能基于普遍的细胞周期缺陷来解释，因为c-myc+/-细胞仅表现出增殖最小程度（20%）的降低。遗传上位性分析表明，c-Myc和Ras通过独立途径发挥作用，这些途径汇聚以调节细胞周期蛋白依赖性激酶抑制剂蛋白p27Kip1的丰度。脱离贴壁培养引发p27的强烈上调，c-Myc表达降低50%显著损害了Ras下调p27的能力。我们提出，Ras和c-Myc信号协同调节细胞周期蛋白D-Cdk4/6复合物的活性：前者通过上调细胞周期蛋白D1的表达，后者通过影响复合物的活性。细胞周期蛋白A的异位表达恢复了c-myc+/-细胞的转化潜能，表明它是该途径中的下游遗传成分。从治疗角度来看，有趣的是，尽管转化似乎对c-Myc表达水平非常敏感，但更大程度的降低可以耐受而不会引起任何显著的细胞周期缺陷。
