Induction of cell proliferation in quiescent NIH 3T3 cells by oncogenic c-Raf-1
Induction of cell proliferation in quiescent NIH 3T3 cells by oncogenic c-Raf-1
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DOI：
10.1128/mcb.17.5.2576
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发表时间：
1997-05-01
影响因子：
5.3
通讯作者：
Rapp, UR
中科院分区：
文献类型：
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作者：
Kerkhoff, E;Rapp, UR
c-Raf-1激酶被不同的促有丝分裂刺激激活，并且已被证明是生长因子应答的重要介质。c-Raf-1激酶的催化结构域与雌激素受体的激素结合结构域（Delta Raf-ER）的融合提供了致癌激活的c-Raf-1的酶调节形式。我们建立了稳定表达c-Raf-1缺失突变体-雌激素受体融合蛋白（c-Raf-1-BxB-ER（TM））的NIH 3 T3细胞（N-BxB-ER（TM）细胞）。这些细胞的转化形态依赖于雌激素拮抗剂3-羟基他诺昔芬的存在。向N-BxB-ER（TM）细胞中加入4-羟基他莫昔芬，通过密度或血清饥饿条款使这些细胞重新进入细胞增殖。在融合细胞中致癌c-Raf-1蛋白激活后24小时，细胞数量明显增加。在血清饥饿的细胞中，增殖的开始进一步延迟，大约需要48小时。在这两种情况下，致癌c-Raf-1诱导的细胞增殖的增殖反应弱于血清介导的增殖反应，不会导致指数生长。这反映在晚期S期和G（2）/M期特异性细胞周期蛋白B蛋白的表达显著降低，以及在G（1）/S转换时诱导的细胞周期蛋白A蛋白的表达略低。c-Raf-1的致癌激活诱导肝素结合表皮生长因子的表达。Jnk 1激酶在自分泌生长因子的作用下被激活。Jnk 1激酶活性的动力学被延迟，并且发生在我们还检测到DNA合成和S期特异性细胞周期蛋白A蛋白表达的时间。这一发现表明，c-Raf-1蛋白的致癌激活可以触发进入细胞周期，而不需要自分泌生长因子环的作用。在所有测试的培养条件下，c-Raf-1-BxB-ER（TM）蛋白的激活导致高水平的细胞周期蛋白D1蛋白的积累和p27（Kip 1）细胞周期蛋白依赖性激酶抑制剂的抑制。
The c-Raf-1 kinase is activated by different mitogenic stimuli and has been shown to be an important mediator of growth factor responses. Fusion of the catalytic domain of the c-Raf-1 kinase with the hormone binding domain of the estrogen receptor (Delta Raf-ER) provides a hormone-regulated form of oncogenic activated c-Raf-1. We have established NIH 3T3 cells stably expressing a c-Raf-1 deletion mutant-estrogen receptor fusion protein (c-Raf-1-BxB-ER(TM)) (N-BxB-ER(TM) cells). The transformed morphology of these cells is dependent on the presence of the estrogen antagonist 3-hydroxytarnoxifen Addition of 4-hydroxytamoxifen to N-BxB-ER(TM) cells arrested by density or serum starvation clauses reentry of these cells into cell proliferation. Increases in the cell number are obvious by 24 h after activation of the oncogenic c-Raf-1 protein in confluent cells. The onset of proliferation in serum-starved cells is further delayed and takes about 48 h. In both cases, the proliferative response of the oncogenic c-Raf-1-induced cell proliferation is weaker than the one mediated by serum and does not lead to exponential growth. This is reflected in a markedly lower expression of the late-S- and G(2)/M-phase-specific cyclin B protein and a slightly lower expression of the cyclin A protein being induced at the G(1)/S transition. Oncogenic activation of c-Raf-1 induces the expression of the heparin binding epidermal growth factor. The Jnk1 kinase is putatively activated by the action of the autocrine growth factor. The kinetics of Jnk1 kinase activity is delayed and occurs by a time when we also detect DNA synthesis and the expression of the S-phase-specific cyclin A protein. This finding indicates that oncogenic activation of the c-Raf-1 protein can trigger the entry into the cell cycle without the action of the autocrine growth factor loop. The activation of the c-Raf-1-BxB-ER(TM) protein leads to an accumulation of high levels of cyclin D1 protein and a repression of the p27(Kip1) cyclin-dependent kinase inhibitor under all culture conditions tested.