Matsuguchi T, Kraft A S
Division of Medical Oncology, University of Colorado Health Science Center, Denver 80262, USA.
Oncogene. 1998 Nov 26;17(21):2701-9. doi: 10.1038/sj.onc.1202201.
To examine the biochemical pathways by which activated Ha-Ras(G12V) (Ha-RasV12) induces factor-independent growth of myeloid cells, Ha-Ras effector loop mutations, including Y40C, T35S, and E37G, were analysed in a mouse factor-dependent myeloid cell line, WT19. Expression of a single effector loop mutant, Ha-Ras(G12V, Y40C) (Ha-RasV12C40), inhibited factor-withdrawal apoptosis, suggesting that activation of the phosphatidylinositol 3'-kinase (Pl3K) pathway is essential to prevent cell death. Neither Ha-Ras (G12V, T35S) (Ha-RasV12S35), which activates the Rafl signaling pathway, nor Ha-Ras(G12V, E37G) (Ha-RasV12G37), which stimulates the RalGDS pathway, did not have significant effects on factor-withdrawal apoptosis of myeloid cells. Although Ha-RasV12C40 inhibited apoptosis, it did not stimulate entry into the cell cycle. Cell lines containing the combination of Ha-RasV12G37 and Ha-RasV12C40 were capable of factor-independent cell growth, while expression of the other combinations of the Ha-Ras effector mutants were not. The combined expression of Bcl-2 and Ha-RasV12G37 was not sufficient to stimulate factor independent growth, suggesting that Ha-RasV12C40 activates additional signals, besides blocking apoptosis, which are critical for factor-independent growth of myeloid cells. In factor-starved myeloid cells, inducible expression of Ha-RasV12G37 results in decreased level of p27Kip1 protein, a cyclin-dependent kinase inhibitor (CKI). These data suggest that the factor-independent growth of myeloid cells requires the activation of at least two pathways, one inhibiting factor-withdrawal apoptosis, and another causing cell cycle progression.
为了研究活化的Ha-Ras(G12V)（Ha-RasV12）诱导髓系细胞因子非依赖性生长的生化途径，我们在小鼠因子依赖性髓系细胞系WT19中分析了Ha-Ras效应环突变，包括Y40C、T35S和E37G。单个效应环突变体Ha-Ras(G12V, Y40C)（Ha-RasV12C40）的表达抑制了因子撤除诱导的凋亡，这表明磷脂酰肌醇3'-激酶（PI3K）途径的激活对于防止细胞死亡至关重要。激活Raf1信号通路的Ha-Ras(G12V, T35S)（Ha-RasV12S35）和刺激RalGDS途径的Ha-Ras(G12V, E37G)（Ha-RasV12G37）对髓系细胞因子撤除诱导的凋亡均无显著影响。虽然Ha-RasV12C40抑制了凋亡，但它并未刺激细胞进入细胞周期。含有Ha-RasV12G37和Ha-RasV12C40组合的细胞系能够进行因子非依赖性细胞生长，而Ha-Ras效应突变体的其他组合表达则不能。Bcl-2和Ha-RasV12G37的联合表达不足以刺激因子非依赖性生长，这表明Ha-RasV12C40除了阻断凋亡外，还激活了其他对于髓系细胞因子非依赖性生长至关重要的信号。在因子饥饿的髓系细胞中，Ha-RasV12G37的诱导表达导致细胞周期蛋白依赖性激酶抑制剂（CKI）p27Kip1蛋白水平降低。这些数据表明，髓系细胞的因子非依赖性生长需要至少两条途径的激活，一条抑制因子撤除诱导的凋亡，另一条促使细胞周期进展。
