Detection of caspase activation in situ by fluorochrome-labeled caspase inhibitors
Detection of caspase activation in situ by fluorochrome-labeled caspase inhibitors
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DOI：
10.2144/01313pf01
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发表时间：
2001-09-01
期刊：
影响因子：
2.7
通讯作者：
Phelps, DJ
中科院分区：
文献类型：
--
作者：
Amstad, PA;Yu, G;Phelps, DJ
细胞凋亡依赖于一组被称为半胱天冬酶的蛋白水解酶的激活。Caspase的激活可以通过使用Caspase特异性抗体进行免疫印迹检测，或者使用在Caspase切割后变成荧光的前荧光底物来检测Caspase的活性。这些方法大多需要制备call提取物，因此不适用于活细胞内活性半胱天冬酶的检测。利用FAM-VAD-FMK，我们开发了一种简单灵敏的检测活细胞中caspase活性的方法。FAM- vad - fmk是一种羧基荧光素（FAM）衍生物，由苯氧羰基缬氨酸-丙氨酸-天冬氨酸-氟甲基酮（zVAD-FMK）合成，是一种专利的广谱半胱天冬酶抑制剂。FAM-VAD-FMK进入细胞并不可逆地与活化的半胱天蛋白酶结合。结合FAM-VAD-FMK的细胞可以通过流式细胞术、荧光显微镜、使用FAM-VAD-FMK，我们测量了活的非贴壁细胞和贴壁细胞中caspase的激活。我们发现FAM-VAD-FMK标记了喜树碱或星孢素治疗后发生凋亡的Jurkat和HeLa细胞。未受刺激的阴性对照细胞未染色。通用caspase抑制剂zVAD-FMK预处理可阻断诱导Jurkat和HeLa细胞的caspase特异性染色。FAM-VAD-FMK预处理staurosporine诱导的Jurkat细胞，抑制caspase-3、-6、-7的亲和标记，阻断caspase特异性细胞染色，抑制细胞凋亡。相比之下，荧光对照抑制剂FAM-FA-FMK没有作用。在96孔板上测量caspase激活显示，与对照细胞相比，staurosporine处理细胞的fam荧光增加了3至5倍。总之，我们表明FAM-VAD-FMK是一种多功能和特异性的工具，用于检测活细胞中活化的半胱天蛋白酶。
Apoptosis is dependent on the activation of a group of proteolytic enzymes called caspases. Caspase activation can be defected by immunoblotting using caspase-specific antibodies or by caspase activity measurement employing pro-fluorescent substrates that become fluorescent upon cleavage by the caspase. Most of these methods require the preparation of call extracts and, therefore, are not suitable for the detection of active caspases within the living cell. Using FAM-VAD-FMK, we have developed a simple and sensitive assay for the defection of caspase activity in living cells. FAM-VAD-FMK is a carboxyfluorescein (FAM) derivative of benzyloxycarbonyl-valine-alanine-aspartic acid-fluoromethyl ketone (zVAD-FMK), which is a patent broad-spectrum inhibitor of caspases. FAM-VAD-FMK enters the cell and irreversibly binds to activated caspases. Cells containing bound FAM-VAD-FMK can be analyzed by flow cytometry, fluorescence microscopy,. or a fluorescence plate reader Using FAM-VAD-FMK, we have measured caspase activation in live non-adherent and adherent cells. We show that FAM-VAD-FMK labeled Jurkat and HeLa cells that had undergone apoptosis following treatment with camptothecin or staurosporine. Non-stimulated negative control cells were not stained. Pretreatment with the general caspase inhibitor zVAD-FMK blocked caspase-specific staining in induced Jurkat and HeLa cells. Pretreatment of staurosporine-induced Jurkat cells with FAM-VAD-FMK inhibited affinity labeling of caspase-3, -6, and -7, blocked caspase-specific cell staining, and led to the inhibition of apoptosis. In contrast, the fluorescent control inhibitor FAM-FA-FMK had no effect. Measurement of caspase activation in 96-well plates showed a 3- to 5-fold increase in FAM-fluorescence in staurosporine-treated cells compared to control cells. In summary, we show that FAM-VAD-FMK is a versatile and specific tool for detecting activated caspase, in living cells.