NF-κB inhibitor sesquiterpene parthenolide induces concurrently atypical apoptosis and cell necrosis:: Difficulties in identification of dead cells in such cultures
NF-κB inhibitor sesquiterpene parthenolide induces concurrently atypical apoptosis and cell necrosis:: Difficulties in identification of dead cells in such cultures
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DOI：
10.1002/cyto.a.10057
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发表时间：
2003-08-01
期刊：
影响因子：
3.7
通讯作者：
Darzynkiewicz, Z
中科院分区：
文献类型：
--
作者：
Pozarowski, P;Halicka, DH;Darzynkiewicz, Z
背景：细胞凋亡和坏死（“意外细胞死亡”）是细胞死亡的不同模式。通常区分凋亡细胞与坏死细胞的特征是质膜完整性的保持，这通过前者细胞在一定时间长度内排除阳离子染料如碘化丙啶（PI）的能力来反映。在坏死过程中，质膜迅速破裂，坏死细胞被PI强烈染色。在研究抗炎倍半萜小白菊（PRT）的细胞生长抑制作用时，我们注意到，在同一培养物中，细胞在凋亡的同时因坏死而死亡。此外，由于细胞凋亡是非典型的，反映在质膜完整性的快速丧失，因此很难根据这一特征区分凋亡和坏死细胞。（a）用PRT处理的HL-60细胞进行半胱天冬酶活化的分析，使用检测活化的（裂解的）半胱天冬酶-3的抗体，（B）通过结合荧光染料标记的半胱天冬酶抑制剂FAM-VAD-FMK结合PI排除试验鉴定凋亡细胞，和（c）用吖啶橙子（AO）对细胞DNA和RNA进行差异染色。凋亡细胞的特征在于（a）免疫细胞化学检测的半胱天冬酶-3活化和（B）FAM-VAD-FMK的结合，随后或同时（c）排除PI的能力丧失，（d）DNA含量的不足，和（e）RNA含量的相对小的变化。坏死细胞显示（a）没有半胱天冬酶-3活化的证据，（B）没有FAM-VAD-FMK的结合，（c）不能排除PI，（d）RNA的快速损失，和（e）不变的DNA含量。凋亡细胞与坏死细胞的鉴别可能基于半胱天冬酶-3活化的证据，用FAM-VAD-FMK结合PI标记或用AO对细胞DNA和RNA进行差异染色。这些数据表明，质膜似乎是PRT的目标之一，因为它的完整性在细胞死亡过程中很早就丧失了，这反映在非典型凋亡和原发性坏死（膜溶解）。(C)2003 Wiley-Liss，Inc.
Background: Apoptosis and necrosis ("accidental cell death") are distinct modes of cell death. The feature that often distinguishes apoptotic from necrotic cells is preservation of the plasma membrane integrity, reflected by ability of the former cells to exclude cationic dyes such as propidium iodide (PI) for a certain length of time. During necrosis, the plasma membrane is rapidly ruptured and necrotic cells stain intensely with PI. While studying cytostatic effects of the anti-inflammatory sesquiterpene parthenolide (PRT), we have noticed that, concurrent with apoptosis, the cells were dying by necrosis in the same cultures. Furthermore, because apoptosis was atypical, reflected by rapid loss of plasma membrane integrity, it was difficult to distinguish apoptotic from necrotic cells based on this feature.Methods: Three methods were used to distinguish apoptosis from necrosis: (a) HL-60 cells treated with PRT were subjected to analysis of caspases activation using antibody that detects activated (cleaved) caspase-3, (b) apoptotic cells were identified by binding of fluorochrome-labeled inhibitor of caspases FAM-VAD-FMK combined with the PI exclusion assay, and (c) cellular DNA and RNA were differentially stained with acridine orange (AO).Results: Apoptotic cells were characterized by (a) caspase-3 activation detected immunocytochemically and (b) binding of FAM-VAD-FMK followed by or concurrent with (c) loss of ability to exclude PI, (d) deficit in DNA content, and (e) relatively little changed RNA content. Necrotic cells showed (a) no evidence of caspase-3 activation, (b) no binding of FAM-VAD-FMK, (c) inability to exclude PI, (d) rapid loss of RNA, and (e) unchanged DNA content.Conclusions: identification of apoptotic cells versus necrotic cells was possible either based on the evidence of caspase-3 activation, by labeling with FAM-VAD-FMK combined with PI or by differential staining of cellular DNA and RNA with AO. The data indicate that plasma membrane appears to be one of the targets of PRT, because its integrity is lost very early during cell death, which is reflected by atypical apoptosis and by primary necrosis (lysis of the membrane). (C) 2003 Wiley-Liss, Inc.