Imaging CXCR4 signaling with firefly luciferase complementation
Imaging CXCR4 signaling with firefly luciferase complementation
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DOI：
10.1021/ac8005457
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发表时间：
2008-07-15
影响因子：
7.4
通讯作者：
Luker, Gary D.
中科院分区：
文献类型：
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作者：
Luker, Kathryn E.;Gupta, Mudit;Luker, Gary D.
趋化因子及其同源受体在细胞生长、存活以及细胞的组织特异性归巢中具有关键作用。虽然这些功能最初是在正常免疫细胞中发现的，但癌细胞可能会利用趋化因子受体信号来促进原发性肿瘤生长和转移。我们对癌症中趋化因子和趋化因子受体信号传导的了解还很缺乏，尤其是这种信号传导在体内发生的情况。为了理解原发性和转移性疾病的分子调控并开发靶向疗法以提高患者生存率，需要对癌症中趋化因子受体信号传导有新的认识。为了满足这一需求，我们开发了一种分子成像报告基因来研究CXCR4的激活情况，CXCR4是一种在多种常见癌症中调节肿瘤生长和转移的趋化因子受体。该报告系统使用基于萤火虫荧光素酶的蛋白质片段互补分析来检测CXCR4和β - 抑制蛋白分子之间的相互作用，这是趋化因子受体信号传导中常见的早期步骤。在基于细胞的实验中，与趋化因子配体CXCL12（SDF - 1）一起孵育会使生物发光呈剂量依赖性增加，这些蛋白质之间的结合比基础水平诱导增加超过7倍。报告基因的激活可以被CXCR4信号传导的特异性抑制剂阻断。这些报告基因能够在活小鼠体内对CXCR4的激活和抑制进行成像。总体而言，这项研究建立了一种新的成像报告基因，用于探究癌症以及由这种趋化因子受体调控的其他疾病中CXCR4的信号传导。
Chemokines and their cognate receptors have key functions in cell growth, survival, and tissue-specific homing of cells. While these functions first were identified in normal immune cells, cancer cells may co-opt chemokine receptor signaling to promote primary tumor growth and metastasis. Our knowledge of signaling by chemokines and chemokine receptors in cancer is lacking, particularly as this signaling occurs in vivo. New insights into chemokine receptor signaling in cancer are needed to understand molecular regulation of primary and metastatic disease and develop targeted therapies to improve patient survival. To meet this need, we have developed a molecular imaging reporter to investigate activation of CXCR4, a chemokine receptor that regulates tumor growth and metastasis in a variety of common cancers. The reporter system uses a firefly luciferase-based protein fragment complementation assay to detect interactions between CXCR4 and beta-arrestin molecules, a common early step in chemokine receptor signaling. In cell-based assays, incubation with the chemokine ligand CXCL12 (SDF-1) produced dose-dependent increases in bioluminescence with > 7-fold induction above basal levels of association between these proteins. Reporter activation could be blocked with specific inhibitors of CXCR4 signaling. These reporters enabled in vivo imaging of CXCR4 activation and inhibition in living mice. Overall, this research establishes a new imaging reporter for probing CXCR4 signaling in cancer and other diseases regulated by this chemokine receptor.