Monitoring NF-kappa B transactivation potential via real-time PCR quantification of I kappa B-alpha gene expression.
Monitoring NF-kappa B transactivation potential via real-time PCR quantification of I kappa B-alpha gene expression.
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DOI：
10.2165/00066982-200307030-00008
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发表时间：
2003-01-01
期刊：
影响因子：
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通讯作者：
Peyron, Jean-Francois
中科院分区：
文献类型：
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作者：
Bottero, Virginie;Imbert, Veronique;Peyron, Jean-Francois
背景：核因子-kappaB是一种重要的转录因子，参与免疫反应的调节以及细胞的增殖和存活。在炎症、神经系统疾病和癌症等多种病理状态中，可以观察到核因子-kappa B的异常和结构性激活。方法和结果：核因子-kappa B转录的终止是通过依赖于核因子-kappaB的I kappa B-α抑制亚单位的合成来实现的。为了定量检测核因子-kappa B的活性，我们用实时定量聚合酶链式反应检测了I-kappa B-αm RNA的表达。用细胞因子肿瘤坏死因子α或佛波酯PMA刺激Jurkat白血病T细胞或HeLa癌细胞的结果与Northern印迹或基因报告分析结果完全一致。这种方法也可以用来评估霍奇金淋巴瘤细胞系中核因子-kappaB的结构性激活。在HeLa细胞中的动力学实验表明，肿瘤坏死因子刺激首先在30分钟内诱导核因子-kappa B DNA结合，然后在30分钟后诱导I-kappa B-α基因转录。去除刺激后的肿瘤坏死因子导致核因子-kappa B DNA结合活性和I-kappa B-αmRNA水平更快地下降。没有检测到I kappa B-αmRNA的积累或稳定，这可能会影响对结果的解释。结论：实时定量聚合酶链式反应检测核因子-kappa B-αm RNA水平是一种快速、灵敏、有效的检测核因子-kappaB转录能力的方法，可用于临床评价核因子-kappaB的状态。
BACKGROUND: Nuclear factor-kappa B (NF-kappa B) is an important transcription factor involved in the regulation of immune responses as well as in cell proliferation and survival. An abnormal and constitutive activation of NF-kappa B is observed in many pathological states as diverse as inflammation, neurological diseases, and cancer.METHODS AND RESULTS: Termination of NF-kappa B transcription is mediated through the NF-kappa B-dependent synthesis of the I kappa B-alpha inhibitory subunit. To quantify NF-kappa B activation we measured by real-time PCR the expression of I kappa B-alpha mRNA. The PCR data perfectly matched the results obtained by Northern blot or gene reporter analysis when Jurkat leukemic T cells or HeLa carcinoma cells were stimulated with various activators of NF-kappa B, such as the cytokine tumor necrosis factor (TNF)-alpha or the phorbol ester PMA. Constitutive NF-kappa B activation in Hodgkin's lymphoma cell line could also be evaluated by this approach. Kinetic experiments in HeLa cells show that TNF stimulation first induced NF-kappa B DNA binding within 30 minutes, followed by I kappa B-alpha gene transcription 30 minutes later. Removal of TNF after stimulation resulted in a faster decrease in both NF-kappa B DNA binding activity and I kappa B-alpha mRNA levels. No accumulation or stabilization of I kappa B-alpha mRNA was detected that could bias interpretation of the results. The sensitivity of the method allowed the detection of NF-kappa B activation in stimulated normal peripheral blood lymphocytes.CONCLUSION: The real-time PCR measure of I kappa B-alpha mRNA levels is a rapid, sensitive, and powerful method to quantify the transcriptional power of NF-kappa B. It can be easily used for clinical evaluation of NF-kappa B status.