Green fluorescent protein tagging of extracellular signal-regulated kinase and p38 pathways reveals novel dynamics of pathway activation during primary and metastatic growth
Green fluorescent protein tagging of extracellular signal-regulated kinase and p38 pathways reveals novel dynamics of pathway activation during primary and metastatic growth
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DOI：
10.1158/0008-5472.can-04-0113
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发表时间：
2004-10-15
期刊：
影响因子：
11.2
通讯作者：
Rosenbaum, SK
中科院分区：
文献类型：
--
作者：
Aguirre-Ghiso, JA;Ossowski, L;Rosenbaum, SK
我们描述了一种新的方法，可以检测体内的原发性和转移性细胞，其中细胞外信号调节激酶（ERK）或p38途径被激活。我们最近的研究结果表明，ERK和p38激酶分别调节体内决定细胞增殖（高ERK与p38比值）或生长停滞和休眠（低ERK与p38比值）的程序。因此，我们能够使用绿色荧光蛋白（GFP）来反映ERK和p38的活性，从而反映癌细胞的增殖状态。这是通过转染致瘤性T-HEp3和HT1080细胞以及休眠的D-HEp3细胞来完成的，质粒编码Elk-GAL4或CHOP-GAL4融合蛋白，当它们分别被ERK或p38磷酸化时，会激活gfp报告基因。这些细胞的命运在培养、原发部位和鸡胚胎和裸鼠的自发转移中进行了检查。在培养中，GFP水平与先前建立的ERK或p38激活水平成正比。相比之下，在体内接种的前24小时内，致瘤细胞和休眠细胞都强烈激活了p38途径。然而，在致瘤细胞中，p38活性被迅速沉默，纠正了ERK/p38失衡，并在整个肿瘤生长期间促进了高ERK活性。相反，在休眠细胞形成的小结节中，ERK活性水平显著降低，而p38活性保持高水平。ERK的强烈激活在转移部位是明显的，而p38的激活在这个解剖位置也是沉默的。这些结果表明，使用该报告系统可以直接测量癌细胞对微环境的反应，并且只有具有增殖能力的细胞才能快速适应ERK和p38信号以实现增殖成功。这种方法允许分离和进一步表征转移细胞与特定的信号特征表明其表型。
We describe a novel approach that allows detection of primary and metastatic cells in vivo in which either the extracellular signal-regulated kinase (ERK) or the p38 pathway is activated. Our recent findings showed that ERK and p38 kinases regulate, respectively, programs dictating cell proliferation (high ERK-to-p38 ratio) or growth arrest and dormancy (low ERK-to-p38 ratio) in vivo. Thus, we were able to use green fluorescent protein (GFP) to reflect ERK and p38 activities and, consequently, the proliferative state of cancer cells. This was accomplished by transfecting tumorigenic T-HEp3 and HT1080 cells, and dormant D-HEp3 cells, with plasmids coding for Elk-GAL4 or CHOP-GAL4 fusion proteins that, when phosphorylated by either ERK or p38, respectively, transactivated a GFP-reporter gene. The fate of these cells was examined in culture, in primary sites, and in spontaneous metastasis in chick embryos and nude mice. In culture GFP level was directly proportional to the previously established levels of ERK or p38 activation. In contrast, during the first 24 hours of in vivo inoculation, both the tumorigenic and the dormant cells strongly activated the p38 pathway. However, in the tumorigenic cells, p38 activity was rapidly silenced, correcting the ERK/p38 imbalance and contributing to high ERK activity throughout the entire period of tumor growth. In contrast, in the small nodules formed by dormant cells, the level of ERK activity was dramatically reduced, whereas p38 activity remained high. Strong activation of ERK was evident in metastatic sites, whereas p38 activation was silenced in this anatomic location as well. These results show that it is possible to directly measure cancer cell response to microenvironment with this reporter system and that only proliferation-competent cells have the ability to rapidly adapt ERK and p38 signaling for proliferative success. This approach allows isolation and further characterization of metastatic cells with specific signaling signatures indicative of their phenotypes.