Pozarowski P, Huang X, Halicka D H, Lee B, Johnson G, Darzynkiewicz Z
Brander Cancer Research Institute, New York Medical College, Valhalla, New York, USA.
Cytometry A. 2003 Sep;55(1):50-60. doi: 10.1002/cyto.a.10074.
Fluorochrome-labeled inhibitors of caspases (FLICA, e.g., FAM-VAD-FMK, FITC-VAD-FMK) have been designed as affinity labels of the enzyme active center of caspases Their binding by apoptotic cells was interpreted as reflecting activation of caspases. We have recently observed, however, that their binding is more complex and may involve additional mechanisms. Our goal in this study was to clarify the ongoing utility of these probes.
Apoptosis of HL-60, Jurkat, MCF-7 and T-24 cells was induced by the DNA topoisomerase I inhibitor, topotecan, or by oxidative stress (H(2)O(2)). Lymphocytes were induced by their mitogenic activation. Using multiparameter laser scanning and flow cytometry analysis, the correlation between FLICA binding and the number of known apoptotic indicators was examined. These included: collapse of the mitochondrial transmembrane potential; activation of caspase-3 (detected immunocytochemically); binding of annexin V; chromatin condensation; the presence of DNA strand breaks; and loss of plasma membrane capability to exclude propidium iodide (PI). FLICA binding specificity was tested by pretreatment with z-VAD-FMK or z-DEVD-FMK.
FLICA binding was subsequent to the collapse of mitochondrial transmembrane potential, nearly concurrent with caspase-3 activation, and preceded annexin V binding, chromatin condensation, DNA fragmentation and loss of plasma membrane integrity. The predominant portion of FAM-VAD-FMK, FITC-VAD-FMK or FAM-DEVD-FMK binding to apoptotic cells could not be inhibited by z-VAD-FMK or z-DEVD-FMK, respectively, when the unlabeled inhibitors were added post-induction of apoptosis.
FLICA are specific and convenient to use markers of apoptotic cells and they detect very early events of apoptosis associated with caspases activation. Assays that combine their binding with either the loss of mitochondrial potential or with exclusion of PI as a probe of plasma membrane integrity, distinguish sequential stages of apoptosis and are particularly useful to differentiate between apoptosis and necrosis. Our results conform with the published data that unlabeled caspase inhibitors, when added after induction of apoptosis, cannot prevent activation of caspases detected by binding of biotinylated inhibitors or by cleavage of fluorogenic substrates. While FLICA binding by apoptotic cells most likely is a consequence of caspase activation, these binding events may also involve other or additional mechanisms than simply their specific attachment to the active enzyme centers of caspases.
荧光染料标记的半胱天冬酶抑制剂（FLICA，例如FAM-VAD-FMK、FITC-VAD-FMK）已被设计为半胱天冬酶酶活性中心的亲和标记物。凋亡细胞对它们的结合被解释为反映了半胱天冬酶的激活。然而，我们最近观察到，它们的结合更为复杂，可能涉及其他机制。我们在本研究中的目标是阐明这些探针的持续实用性。
用DNA拓扑异构酶I抑制剂拓扑替康或氧化应激（H₂O₂）诱导HL-60、Jurkat、MCF-7和T-24细胞凋亡。通过有丝分裂原激活诱导淋巴细胞凋亡。使用多参数激光扫描和流式细胞术分析，检测FLICA结合与已知凋亡指标数量之间的相关性。这些指标包括：线粒体跨膜电位的崩溃；半胱天冬酶-3的激活（通过免疫细胞化学检测）；膜联蛋白V的结合；染色质浓缩；DNA链断裂的存在；以及质膜排除碘化丙啶（PI）能力的丧失。通过用z-VAD-FMK或z-DEVD-FMK预处理来测试FLICA结合的特异性。
FLICA结合发生在线粒体跨膜电位崩溃之后，与半胱天冬酶-3激活几乎同时发生，且先于膜联蛋白V结合、染色质浓缩、DNA片段化和质膜完整性丧失。当在凋亡诱导后添加未标记的抑制剂时，FAM-VAD-FMK、FITC-VAD-FMK或FAM-DEVD-FMK与凋亡细胞结合的主要部分分别不能被z-VAD-FMK或z-DEVD-FMK抑制。
FLICA是凋亡细胞特异性且方便使用的标记物，它们能检测与半胱天冬酶激活相关的非常早期的凋亡事件。将它们的结合与线粒体电位丧失或PI排除作为质膜完整性探针相结合的检测方法，可区分凋亡的连续阶段，对区分凋亡和坏死特别有用。我们的结果与已发表的数据一致，即未标记的半胱天冬酶抑制剂在凋亡诱导后添加时，不能阻止通过生物素化抑制剂结合或荧光底物切割检测到的半胱天冬酶激活。虽然凋亡细胞对FLICA的结合很可能是半胱天冬酶激活的结果，但这些结合事件可能还涉及其他或额外的机制，而不仅仅是它们与半胱天冬酶活性酶中心的特异性结合。
